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Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONApoptosis (gene-directed cell...Relationship of intracellular...Implications for conditions of...Biochemical mechanisms of zinc...Future prioritiesREFERENCES |
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KEY WORDS: • zinc • apoptosis • caspase • disease • Zinquin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONApoptosis (gene-directed cell...Relationship of intracellular...Implications for conditions of...Biochemical mechanisms of zinc...Future prioritiesREFERENCES |
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).
Another fundamental cellular process that may be regulated by Zn is the
cell suicide process of apoptosis (also known as gene-directed cell
death or, less correctly, as programmed cell death) (reviewed in Wyllie 1997
) (for detailed reviews on the earlier reports relating Zn and
apoptosis, see Sunderman 1995
, Zalewski et al. 1996
and Zalewski and Forbes 1993
). This review
focuses on more recent studies and attempts to relate Zn to the current
concepts of the regulation of apoptosis. It also takes a critical look
at some of the interpretations that have emerged and attempts to
identify some of the gaps in our understanding of Zn and apoptosis.
Any consideration of the cellular biology of Zn and its role in
regulation of apoptosis and other growth processes must recognize, by
necessity, the diverse functions of this metal, which extend from
highly stable structural roles within folded protein domains to
transient interactions within cellular signaling pathways. These
diverse functions can be classified broadly into those dependent on a
largely fixed pool of cellular Zn (e.g., stoichiometric amounts of Zn
that are tightly bound within the tertiary protein structure of
metalloenzymes and poorly exchangeable) and the more dynamic, labile Zn
pools that are subject to ionic fluxes and readily influenced by Zn
deprivation or supplementation (Frederickson 1989
,
Vallee and Falchuk 1993
, Zalewski et al. 1993
). It is this labile pool that we focus on in the context
of the regulation of apoptosis. To visualize and quantify this pool, we
used a sulfonamidoquinoline-based UV-excitable Zn fluorophore,
Zinquin. Studies with Zinquin, and the related fluorophore TS-Q,
have revealed relatively slow (time scale of hours) Zn fluxes
associated with a number of physiological and pathological processes,
including gene expression, secretion, mitosis, apoptosis,
spermatogenesis, fertilization, early embryonic development and
inflammation (Frederickson 1989
, Zalewski et al. 1993
, 1994a
, 1994b
and
1996
).
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Apoptosis (gene-directed cell death) |
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TOPABSTRACTINTRODUCTIONApoptosis (gene-directed cell...Relationship of intracellular...Implications for conditions of...Biochemical mechanisms of zinc...Future prioritiesREFERENCES |
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).
Apoptosis is prominent during embryonic and fetal development and in
the immune response, among other processes, as well as being a target
for growth factors, hormones and cytokines; in addition, it is a major
mechanism of cell death in the body in response to toxic agents (e.g.,
x-irradiation), and its dysregulation (insufficient or excessive) is
central to pathogenic mechanisms in many diseases (e.g.,
neurodegenerative disorders, acquired immune deficiency syndrome,
autoimmune disease and malignancy) (Cohen and Duke 1993
,
Kerr et al. 1987
, Wyllie 1997
). As such,
apoptosis has become a novel therapeutic target in medicine, and the
factors regulating its induction and execution are being intensively
studied.
Apoptosis occurs in two phases, consisting of (1) the
biochemical signaling pathways that commit a cell to apoptosis and
(2) the executional phase characterized by stereotypical
morphological changes leading to cell death. Apoptosis results from the
interaction between the initiating stimuli, which can be either
physiological or injurious to the cell, and the factors determining the
susceptibility of the cell to apoptosis. The input signaling pathways
are diverse and may originate from plasma membrane receptors, newly
transcribed gene products or disturbance of the microtubular
cytoskeleton (Wyllie 1997
). These converge onto a
central pathway masterminded by the caspases and regulated by the
proapoptotic Bax-like and antiapoptotic Bcl-2–like family of
mitochondrial membrane proteins (Thornberry and Lazebnik 1998
, Wyllie 1997
). Members of the caspase
family share two key features: they require an aspartic acid at the
cleavage site, and they are all synthesized as proenzymes, which are
activated by cleavage by other caspases, resulting in a cascade of
proteolytic events (Thornberry and Lazebnik 1998
). The
14 members of the caspase family are subdivided into the executioner
caspases, which cleave particular substrates to commit the cell to
irreversible cell death, and the initiator caspases, which act upstream
to relay apoptosis-inducing signals by proteolytically activating
proenzyme forms of the executioner caspases. The best studied of the
executioner caspases is caspase-3, which cleaves proteins containing
the consensus motif DXXD (where X is any amino acid). Substrates
include the cell cycle regulator p21Waf1/Cip1
(Levkau et al. 1998
) and the Ca/Mg-dependent
endonuclease, caspase-activated deoxyribonuclease, leading to
activation of a Ca/Mg-dependent endonuclease that cuts DNA into
nucleosomal fragments, recognized as a "DNA ladder" on gels
(Janicke et al. 1998
). The other major executioner
caspase is caspase-6, which cleaves the nuclear lamin scaffold
proteins, resulting in collapse of the nucleus as well as participating
in the proteolytic activation of caspase-3 (Srinivasula et al. 1996
, Takahashi et al. 1996
).
Cells entering death via apoptosis undergo a distinct set of structural
changes that are consistent throughout all cell types (Kerr et al. 1987
). These include the separation of the dying cell from
its neighbors, loss of microvilli, blebbing of the membrane,
condensation of cytoplasm, increased cell density and the compaction
and segregation of the nuclear chromatin to form dense masses
underlying the nuclear membrane. Condensation of the chromatin
commences around the periphery of the nucleus and later involves most
of the nucleus. This is followed by nuclear fragmentation and the
budding of the cell to produce membrane-bound apoptopic bodies that
are shed into luminal cavities or phagocytosed. The formation of
apoptotic bodies keeps the dying cell out of direct contact of the
remaining healthy tissue. This contrasts to the situation in necrosis,
where cell death and lysis follow physical, chemical or osmotic damage,
liberating potentially toxic cytoplasmic enzymes onto the dying cell’s
neighbors as well as chemotactic factors that may initiate a local
inflammatory reaction (Kerr et al. 1987
).
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Relationship of intracellular labile zinc to apoptosis |
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TOPABSTRACTINTRODUCTIONApoptosis (gene-directed cell...Relationship of intracellular...Implications for conditions of...Biochemical mechanisms of zinc...Future prioritiesREFERENCES |
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Zinc deprivation.
Localized or systemic tissue Zn deficiency can be induced by
malnutrition or the administration of metal chelators. It can also
arise in a number of pathophysiological states, including
malabsorption, increased Zn requirements (e.g., in pregnancy, lactation
and severe burns), increased Zn loss (e.g., zincuria, sweating), heavy
metal poisoning in which Zn is displaced by the metal, aging and
disease (e.g., diabetes mellitus and Down’s syndrome) (Solomons 1988
, Vallee and Falchuk 1993
).
Although systematic studies of apoptosis in Zn-deficient animals
are still lacking, it is clear that the frequency of apoptotic cells is
increased markedly in certain tissues and organs, including the
intestinal and retinal pigment epithelium, skin, thymic lymphocytes,
testis and pancreatic acinar cells (reviewed in Duvall and Wyllie 1986,
Sunderman 1995
, Zalewski and Forbes 1993
). Apoptosis is also markedly increased in
conjunction with congenital abnormalities in fetal rats borne by
Zn-deficient dams. This was emphasized in the neuroepithelium,
where excessive apoptosis interfered with neural tube closure
(Record et al. 1985
). Rogers et al. (1995)
concluded that apoptotic embryonal cell death,
particularly in the neural crest cells, could arise within 4 d of
maternal Zn deficiency. To what extent does increased apoptosis also
occur in Zn-deficient humans? The answer to this question is not
known, largely because of limited access to tissues for analysis. One
approach would be to study the susceptibility of neutrophils to
apoptosis in vivo because it is known that neutrophil Zn levels decline
in even mildly Zn-deficient humans (Pai and Prasad 1988
, Prasad et al. 1993
). Interestingly,
peripheral leukocytes of patients with Down’s syndrome, a disease
associated with Zn deficiency, had a greatly increased frequency of
cells with DNA nicks, which is thought to be an early stage of
apoptosis; these cells were substantially decreased after Zn
supplementation for 6 mo (Antonucci et al. 1997
).
Unclear, however, is whether the appearance of these cells was simply
the result of reduced DNA repair rather than the promotion of
apoptosis.
Increased apoptosis in vivo may be a direct consequence of a decrease
in intracellular Zn concentration
([Zn2+]i) or indirectly
via some other change. This issue has received some attention in the
context of the involution of the thymus in Zn-deficient animals,
where it was concluded that at least in part, the increased apoptosis
of thymocytes was due to excessive levels of circulating
glucocorticoids triggered by a Zn deficiency–associated stress
response (Fraker and Telford 1997
). However, the
evidence from in vitro studies indicates that apoptosis can result
directly from a decline in intracellular Zn within the same cells.
Numerous in vitro studies have shown a direct stimulatory effect on
apoptosis in cells whose levels of Zn have been depleted. Thus,
apoptosis was induced in various types of cells when cultured in a
Zn-free medium (Martin et al. 1991
) or Zn depleted
by membrane-permeant Zn chelators [e.g.,
N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN)
or phenanthroline] (Treves et al. 1994
, Zalewski et al. 1991
,1993
). Zn deficiency–induced
apoptosis has all of the major features of apoptosis (DNA
fragmentation, caspase activation, nuclear fragmentation, chromatin
condensation and apoptotic body formation). However, in some cells
(e.g., T-cell leukemic Molt-3 cells), Zn deprivation resulted in
necrosis (Martin et al. 1991
).
In addition to a direct effect of Zn depletion on induction of
apoptosis, there was a potent synergy between Zn depletion and
suboptimal concentrations of other apoptotic inducers. Phenanthroline,
at concentrations that decreased intracellular Zn, greatly potentiated
hydroxyurea-mediated cytotoxicity of human myeloid leukemia cells
(Kamath et al. 1989
), whereas TPEN potentiated apoptosis
of lymphoid cells (Zalewski et al. 1993
). Zn deficiency
in vivo may also increase the sensitivity of animals to toxins. For
example, colchicine was more toxic to duodenal cells in
Zn-deficient rats than in normal rats (Dinsdale and Williams 1977
).
Zinc supplementation.
Changes in the susceptibility of cells to undergo apoptosis may explain
the altered resistance of animals to certain toxic agents when Zn
intake is increased, particularly because some of these agents induce
apoptosis in vitro and are antagonized by Zn. Thus, small doses of Zn
aspartate, in combination with a thiol reagent, protected against
radiation lethality in mice (Floersheim et al. 1992
),
and Zn blocked sporidesmin-induced apoptosis in macrophages and T
lymphoblasts (Waring et al. 1990
). Zn, when administered
subcutaneously, significantly reduced the increased neuronal cell death
(largely apoptosis) in the hippocampus of gerbils after they were
subjected to transient forebrain ischemia (Matsushita et al. 1996
). In rabbits, topical administration of high
concentrations of Zn to the eye after superficial keratectomy
significantly prevented the loss byapoptosis of the anterior and
stromal keratinocytes (Kuo et al. 1997
). These studies
show that Zn levels in cells can be manipulated in vivo sufficiently to
affect susceptibility toapoptotic agents and toxins. Excessively
high levels of [Zn2+]i
may be toxic, however. Chicks fed a high-Zn diet (2 g/kg) had a
drastic increase in levels of total pancreatic Zn accompanied by high
rates of apoptotic death of pancreatic acinar cells (Markov et al. 1992
). This may indicate either that high
[Zn2+]i can induce
apoptosis or that cells undergoing apoptosis accumulate high
[Zn2+]i.
Zn supplementation studies in vitro support the in vivo findings. In
1976 Chvapil et al.
showed that the inclusion of 10
µmol/L Zn in the cell isolation medium of canine lymphocytes
significantly increased their viability. This effect was accompanied by
an uptake of Zn either on or in the cells. There are now numerous
examples where the presence of extracellular Zn ions (200–5000
µmol/L) suppresses apoptosis-related events and, at least in the
short term, cell death (reviewed in Sunderman 1995
,
Zalewski and Forbes 1993
). The requirement for high
concentrations of extracellular Zn to inhibit apoptosis is probably due
to the relatively poor uptake of Zn across the cell membrane. Thus, a
much lower concentration of Zn (50 µmol/L) sufficed to inhibit
Ca-induced DNA fragmentation in isolated nuclei (Cohen and Duke 1984
), and low extracellular concentrations (5–25
µmol/L) of Zn strongly suppressed internucleosomal DNA fragmentation
and cell death in intact cells when administered in the presence of Zn
ionophores such as pyrithione (Zalewski et al. 1991
).
Intracellular zinc and susceptibility to apoptosis.
Previously, we have shown in lymphoid cells that when the level of
intracellular labile Zn was decreased by treatment with a chelator or
increased by treatment with an ionophore, there was good correlation
between the content of labile Zn and the inhibition of DNA
fragmentation (Zalewski et al. 1993
), suggesting that a
reduction below a threshold concentration in
[Zn2+]i induces
apoptosis. The steepness of the threshold curve indicates that
relatively small changes in labile Zn can cause large changes in the
susceptibility of cells to DNA fragmentation.
However, there are still some major reservations with this model.
First, there remain concerns about the interpretation of the in vitro
Zn supplementation studies, especially where excessively high Zn
concentrations have been used. Unfortunately, this includes most
published studies. Apoptosis is an active, energy-dependent
process, and it will be blocked by excessive cell damage. Fraker and Telford (1997
) have shown that even 100 µmol/L Zn is
toxic for mouse thymocytes, although surprisingly the death was by
apoptosis rather than by necrosis. We have preferred to limit
extracellular Zn to 25 µmol/L and to instead use the ionophore
pyrithione to transport Zn into the cells (Zalewski et al. 1993
). However, even here, at higher concentrations of
pyrithione resulting in excessive Zn overload, there is blebbing of the
cells and detachment from the substratum. The second reservation is
that even though an increase in
[Zn2+]i specifically
suppresses apoptosis-related biochemical events, the cells may
still die in the longer term. The evidence that Zn fails to block cell
death in many systems has been reviewed recently (Fraker and Telford 1997
). An often-cited study is that of
Barbeiri et al. (1992
), who reported that Zn blocked
dexamethasone-induced DNA fragmentation in thymocytes but did not
prevent cell death. However, they used such an extremely high
concentration (5 mmol/L) of Zn that it is scarcely surprising that the
cells died.
There are two separate issues here. First, by suppressing apoptosis, Zn may simply divert cells into necrosis. This is not a trivial concern because necrosis of the cells will lead to a greater inflammatory response than if the cells were to undergo apoptosis and be rapidly cleared. Second, the complete apoptotic program may be regulated by multiple factors (see later). Full cytoprotection may require all of these in addition to Zn.
Why zinc?
In considering the mechanisms by which Zn regulates apoptosis, it is
pertinent to ask why Zn and not other metals? It has been argued that
Zn has assumed, during evolution, a special role in mediating events
associated with nucleic acid synthesis and degradation, particularly
after the development of an oxygen-rich atmosphere (da Silva and Williams 1991
). Zn has numerous chemical properties
advantageous for a role in cytoprotection. It protects macromolecules
(e.g., proteins and DNA) from oxidation and proteolysis, and stabilizes
macromolecular complexes (e.g., microtubules) and subcellular
organelles (e.g., membranes) (Vallee and Falchuk 1993
).
Its affinity for sulfydryl groups coupled with lack of redox activity
enables it to reversibly suppress cysteine-dependent enzymes
(including perhaps one or more of the caspases) without irreversible
damage or inactivation.
Notwithstanding this, related metals in the periodic table may also
play a role because in vivo depletion of Cu in rats led to a marked
increase in apoptosis of the acinar cells (Kishimoto et al. 1994
). On the other hand, Fraker and Telford (1997
) found that supplementation of Cu (and Ni) at varying
concentrations up to 500 µmol/L did not mimic Zn in the suppression
of glucocorticoid inhibition of thymocyte apoptosis. Other metals may
antagonize the role of Zn. Although its precise mechanism of action
remains unclear, Ca is known to activate some proapoptotic enzymes
(e.g., endonucleases, proteases and phospholipases) (McConkey & Orrenius, 1997
), whereas both Cd and Au induce apoptosis (and
necrosis) (Fraker and Telford 1997
).
Changes in intracellular zinc early and late in apoptosis.
Assuming that Zn is a physiological regulator of apoptosis, it is
pertinent to ask what happens to
[Zn2+]i in cells that are
induced to undergo apoptosis? Initially, there may be a decline in
[Zn2+]i. Several
apoptosis-inducing agents cause a decrease in
[Zn2+]i (e.g.,
intracellular Zn was decreased before the induction of apoptosis in
lymphoid cells with dexamethasone or ATP; Treves et al. 1994
). However, during the process of apoptosis,
[Zn2+]i rises as shown by
an intense reaction with Zinquin (Zalewski et al. 1994b
). This did not occur in necrosis. We have proposed that
the new pools of Zinquin-reactive Zn arise as a result of a change
in the redox state of the cell that releases Zn bound to protein via
Zn
S thiolate bonds (Zalewski et al. 1994b
). Because
apoptosis is a process by which the dying cell dismantles itself and
because Zn is a structural building block in many cellular components
(e.g., membrane, cytoskeleton and chromatin) (Vallee and Falchuk 1993
), a release of Zn during apoptosis is perhaps not
surprising. After phagocytosis, Zn may be recycled or excreted from the
body. Whether the release is simply an effect of apoptosis or further
accelerates the process (e.g., by destabilization of the microtubules
and other structures, thereby facilitating action of caspases and
endonucleases) is not known. Alternatively, the increase in
fluorescence may result from an influx of extracellular Zn due to the
loss of membrane integrity or decreased membrane efflux (as discussed
in Cuajungco and Lees 1997
). Mechanisms aside, Zn
fluorophores may prove to be useful reagents in screening for apoptotic
cells, particularly in tissues where the frequency of these cells is
very low.
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Implications for conditions of altered zinc homeostasis |
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TOPABSTRACTINTRODUCTIONApoptosis (gene-directed cell...Relationship of intracellular...Implications for conditions of...Biochemical mechanisms of zinc...Future prioritiesREFERENCES |
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and references
within). To what extent do manifestations of Zn deficiency (e.g.,
growth retardation, immunodeficiency, skin lesions, delayed sexual
maturation and gastrointestinal problems) reflect an increase in
apoptosis in the relevant tissues? Mori et al. (1996
)
proposed a role for increased apoptosis in the formation of vesicular
skin lesions in patients with acquired zinc deficiency but not in the
formation of hyperkeratotic skin lesions. To what extent increased
apoptosis contributes to delayed wound healing in Zn deficiency is not
known, but it may reflect changes in the rates of apoptosis in
epithelial cells and fibroblasts. Certainly the atrophy of tissues in
Zn-deficient animals is likely to be due not only to inhibition of
cell proliferation but also to excessive apoptotic cell death, although
there have been no systematic studies of this to date. If Zn is
required to both promote mitosis (reviewed in Chesters 1989
) and suppress apoptosis, it may serve to coordinate these
two opposing growth-regulatory processes. Enhancement of mitosis
and suppression of apoptosis occur in regenerating tissues, whereas
tissue involution and atrophy result from the simultaneous suppression
of mitosis and enhancement of apoptosis. Cellular Zn uptake is enhanced
by growth factors, hormones and other agents that promote mitosis and
suppress apoptosis (e.g., by epidermal growth factor in thymic
epithelial cells; Coto et al. 1992
). Because cells
require both growth factors and Zn to pass the
G1/S restriction point (a cell cycle decision
point ~2–3 h before the onset of DNA synthesis), after which neither
is required (Chester 1989
), events close to this
restriction point may be critical to the mechanism via which the two
opposing growth regulatory processes are coordinated.
Does Zn play a functional role in development-associated programmed
cell death? The only relevant study is that of Budzik et al. (1982
), who showed that Zn chelation by EDTA mimicked
müllerian-inhibiting substance in causing apoptotic regression of
the müllerian duct in fetal organ cultures and that Zn was the
only metal capable of blocking regression caused by both of these
agents. The role of Zn in development requires further study.
If Zn is a physiological regulator of apoptosis, then there may be
implications for degenerative changes in aging where decreased uptake
and utilization of Zn by cells could contribute to the increased
susceptibility of senescent cells to undergo apoptosis. In aging, Zn is
decreased in the plasma, leukocytes, bone, epidermis, testis and
kidney, associated with reduced activities of Zn-dependent enzymes
and hormones as well as depression of Zn-dependent functions,
including immunity, wound healing, smell and taste acuity (see Prasad et al. 1993 and references within
). Is increased
apoptosis in a Zn-deficient setting a factor in the heightened
susceptibility of T-cells in aged animals to apoptosis
(Aggarwal and Gupta 1998
) or in the excessive apoptosis
accompanying neurodegeneration in Alzheimer’s disease (Anderson et al. 1996
)? In diseases complicated by secondary Zn
deficiency, is there excessive apoptosis? For example, in diabetes
mellitus, does the generalized depletion of intracellular Zn
(Pai and Prasad 1988
) also affect the ß-cells and
render them more susceptible to apoptosis induced by autoimmune
cytotoxic T-cells or by chemical diabetogens? This may be relevant
to the mechanism by which Zn chelators cause permanent diabetes in
experimental animals (Goldberg et al. 1990
).
Finally, is labile Zn increased in some tumors, and if so, is this a
factor in their relative resistance to apoptosis and accelerated
growth? There have been relatively few reported studies of Zn in tumors
and very little study of the relationship between level of Zn and tumor
growth. In the sporadic cases reported so far, less Zn was observed in
some tumors, but more was observed in others. For example, levels of Zn
were reported to be increased 75% in rat colon tumors relative to the
adjacent normal tissue (Song et al. 1993
). However,
cancer of the prostate contained less Zn than normal prostatic tissue
(Byar 1974
). Zn supplementation and Zn deficiency can
have both inhibitory and augmenting effects on tumor formation,
depending on the tumor and host species (Kasprzak and Waalkes 1986
). Some tumor cells and transformed cell lines have
acquired a greatly decreased dependency on extracellular Zn for growth
compared with the normal primary cell cultures (e.g., kidney cells)
(Chesters 1989
).
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Biochemical mechanisms of zinc in apoptosis |
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TOPABSTRACTINTRODUCTIONApoptosis (gene-directed cell...Relationship of intracellular...Implications for conditions of...Biochemical mechanisms of zinc...Future prioritiesREFERENCES |
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Molecular targets of zinc.
Initial interest focused on the Zn-mediated suppression of
Ca/Mg-dependent endonuclease, which causes apoptotic DNA fragmentation
by cutting linker regions between nucleosomes generating a ladder
pattern (Cohen and Duke 1984
). In studies from an
unrelated laboratory, Cohen et al. (1992
) observed that
Zn blocked the transition from the morphological stage in which the
chromatin is condensed around the periphery to that stage in which most
of the nucleus is involved, and they also attributed this to specific
inhibition of the Ca/Mg-dependent endonuclease. It has been proposed
that the exchange of Zn for Ca within the nucleus may initiate the DNA
fragmentation (Duvall and Wyllie 1986
). However, there
remain no studies of whether physiological concentrations of Zn
influence the endonuclease and whether it is directly activated in Zn
deficiency.
The possibility that Zn may suppress a step before activation of the
endonuclease was first shown by Lazebnik et al. (1993
).
Using a cell-free model in which cytosol from cells primed to
undergo apoptosis induce nuclear condensation and DNA fragmentation,
they showed that morphological changes in the nuclei were suppressed by
concentrations of Zn lower than those required to suppress the
fragmentation of DNA and that the target was cytoplasmic rather than
nuclear. Because the active cytosolic factor was subsequently
identified as the protease CPP-32 (renamed caspase-3), it was suggested
that Zn either may block the mechanism by which the inactive
procaspase-3 is processed and thereby activated or may block the
capacity of active caspase-3 to cleave its cellular substrates.
Figure 1
shows that in colon cancers induced to undergo apoptosis by the
addition of the histone deacetylase inhibitor butyrate, supplementation
with Zn (using the Zn ionophore pyrithione plus 25 µmol/L
ZnSO4) suppressed a step before the activation of
caspase-3.
|
), at lower, more
physiological concentrations, it is a selective inhibitor of caspase-6
(also known as Mch-2); complete inhibition was observed at 10 µmol/L
Zn (Stennicke and Salvesen 1997
, Takahashi et al. 1996
). There is as yet no physiochemical explanation for the
selective effects of Zn on this enzyme. Caspase-6 is, to date, the most
sensitive apoptosis-related molecular target of Zn, although it
remains to be determined whether this enzyme is influenced by normal
concentrations of Zn present within its subcellular compartment.
Because caspase-6 is responsible for the cleavage of lamins and
therefore is directly involved in the nuclear membrane dissolution
(Srinivasula et al. 1996
, Takahashi et al. 1996
), Zn should retard these events, although this has not yet
been directly tested. In addition, caspase-6 is known to cleave and
activate the proenzyme form of caspase-3 (Srinivasula et al. 1996
). Therefore, Zn should also retard caspase-3 activation.
We tested the latter using a cell-free system in which the addition
of cytochrome c to the cytosol of healthy cells triggers the
proteolytic conversion of procaspase-3 to the active enzyme. The
addition of 800 nmol/L free Zn blocked the activation of caspase-3 by
50% in this model; there was no effect of Zn when added 90 min after
cytochrome c but before the addition of fluorogenic
caspase-3 substrate, confirming that Zn blocks the process of caspase-3
activation rather than the already activated enzyme (Fig. 1
,
inset). This was also shown by Aiuchi et al. (1998
) with the use of Western blotting to track the
caspase-processing. However, the role of caspase-6 in this model is
not known.
Another highly conserved family of cellular proteins that regulate a
common pathway of apoptosis are the antiapoptotic Bcl-2–like and
proapoptotic Bax-like mitochondrial membrane proteins. The ratio of
Bcl-2–like to Bax-like proteins acts as a cellular rheostat to
determine in part survival or death of cells after an apoptotic
stimulus (Korsmeyer et al. 1993
). In a recent report,
Fukamachi et al. (1998
) showed that Zn supplementation
of cells in vitro increased the Bcl-2/Bax ratio, thereby increasing the
resistance of the cells to apoptosis.
Other potential targets for Zn are the microtubular cytoskeleton, which
is disrupted in both Zn deficiency (Nickolson and Veldstra, 1972
) and apoptosis (Martin and Cotter 1990
),
oxidative stress (a central mechanism in the induction of apoptosis
that may be influenced by the antioxidant properties of Zn)
(Vallee and Falchuk 1993
) and, specifically in
glucocorticoid-induced apoptosis, suppression of glucocorticoid
binding to its cytoplasmic receptors (Fraker and Telford 1997
).
Still unclear is whether each of these candidate molecular targets of Zn reflects physiological, pharmacological or toxicological actions. A criticism with many of the studies to date is that only the effects of Zn supplementation (not depletion) were tested and that very high (supraphysiological concentrations of Zn) were present in the extracellular medium. The activity of caspase-6 and mechanisms involved in the activation of caspase-3 may be exceptions because they were suppressed by much lower concentrations of Zn. The effects of Zn chelators at the molecular level have been largely neglected. In fact, it remains uncertain whether Zn deprivation and Zn supplementation are affecting the same step in the cascade.
What are critical intracellular pools?
Which of the subcellular pools of Zn participate in the suppression of
apoptosis? Potential targets may exist in the nucleus (Ca/Mg
endonuclease, gene promoters), cytosol (caspase-6, metallothionein,
oxyradicals, Ca ions), cytoskeleton (microtubules) and mitochondria
(Bcl-2/Bax). Multiple subcellular pools are probable because in
cell-free models of apoptosis, Zn inhibits apoptosis regardless of
whether it is added to isolated nuclei (Cohen and Duke 1984
) or to cytosol (Lazebnik et al. 1993
). When
intracellular pools of Zn were increased by Zn ionophore under
conditions that suppress apoptosis, there was little increase in
nuclear Zn, except for occasional intense labeling of nucleoli, but
there was a strong increase in cytoplasmic fluorescence that appeared
to be localized within membrane-enclosed vesicles (Fig. 2
). This may indicate that the target or targets are primarily
cytoplasmic (e.g., caspases). To what extent Zn interacts with the
cytoplasmic pools of caspases and their precursors is not known, nor is
it known whether the proenzyme and activated forms of the caspases are
within or associated with Zn-rich vesicles and influenced by Zn
supplementation or Zn deprivation.
|
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Future priorities |
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TOPABSTRACTINTRODUCTIONApoptosis (gene-directed cell...Relationship of intracellular...Implications for conditions of...Biochemical mechanisms of zinc...Future prioritiesREFERENCES |
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A substantial body of evidence now implicates Zn in the regulation of apoptosis in vitro. This evidence includes studies of the effects of Zn deprivation or supplementation on either the spontaneous rate of apoptosis or the susceptibility of cells to the induction of apoptosis by other agents, as well as studies of the effects of Zn on various components of the apoptotic signaling pathway. We now need to obtain further evidence from a variety of models that address the following three hypotheses: (1) that a specific pool or pools of intracellular labile Zn regulate apoptosis; (2) that systemic changes in Zn levels in the body, due to dietary factors, altered physiological states or disease, can sufficiently alter labile intracellular Zn to change cell susceptibility to apoptosis, and (3) that this altered susceptibility to apoptosis contributes to changes in the body. These hypotheses at best remain tenuous at this stage, and it is premature to conclude that Zn is a physiological regulator of apoptosis.
It may be instructive at this stage to compare Zn with the best known
physiological suppressor of apoptosis Bcl-2 (and related members of the
family). There are some interesting parallels between the two factors.
Both are antagonists of a central mechanism in apoptotic cell death and
therefore suppress apoptosis in response to a variety of inducers
acting via diverse pathways. Both inhibitors appear to act on apoptosis
at multiple overlapping sites. Thus, like Zn, Bcl-2 protects cells from
oxidative stress (Korsmeyer et al. 1993
) and directly
suppresses caspase processing (Thornberry and Lazebnik 1998
). Like severely Zn-deficient animals (Solomons 1988
, Vallee and Falchuk 1993
), bcl-2 knockout
mice have stunted growth; immunodeficiency associated with greatly
increased apoptosis within the thymus, sparing only the epithelial
cells and macrophages; and hair hypopigmentation, thought to be due to
an oxidation-related pathology (Korsmeyer et al. 1993
). Finally, feedback mechanisms may ensue to release cells
from suppression by both Zn and Bcl-2 once the cells enter the
apoptotic pathway. In the case of Bcl-2–like proteins, they normally
act to suppress the processing of caspases and restrict entry into
apoptosis, but during apoptosis they become substrates for caspases
(Fujita et al. 1998
). This down-regulation may
accelerate downstream events. Similarly, although Zn may normally
suppress caspase activation in healthy cells, the dramatic changes in
intracellular Zn homeostasis during apoptosis may relieve suppression
and facilitate downstream events such as endonuclease activation.
Whether Zn and Bcl-2 have overlapping, additive or synergistic
functions in control of apoptosis awaits determination.
The next problem will be in distinguishing the physiological,
pharmacological and even toxicological effects of Zn. Characterization
of the intracellular pool or pools of Zn that mediate suppression and a
better understanding of how this pool or these pools interact with the
apoptotic signaling and effector pathways arre urgently needed. By
analogy, the realization that Bcl-2 was largely a mitochondrial
membrane protein led to the discovery of its role in cytochrome
c release from mitochondria and subsequent activation of
caspases. New insights into the compartmentalization of caspase
processing and activation in cells coupled with colocalization studies
of labile Zn and caspases (especially caspase-6) may provide further
clues. How many of the other 14 caspases, identified to date, are as
sensitive to Zn as caspase-6 is not known. Such studies, in association
with site-directed mutagenesis of histidines and nonessential
cysteines in caspase-6, should provide a better understanding of how Zn
inhibits this enzyme. There also is a particular need to determine
whether Zn deprivation in vitro and in vivo directly activates these
suicide enzymes. It is not at all clear whether Zn supplementation and
Zn deprivation affect the same targets in the apoptotic cascade,
especially if there are multiple targets of Zn. Some targets may
already be Zn saturated and therefore will be more influenced by Zn
deficiency than by Zn supplementation. Well-defined cell-free
models of caspase processing and apoptosis now exist (Thornberry and Lazebnik 1998
) that can be used to study the role of Zn
deprivation and supplementation under well-controlled conditions.
An area ripe for study is the influence of Zn supplementation in vivo on enhanced rates of apoptosis in primary and secondary Zn deficiency states. If Zn-related apoptosis is increased in Alzheimer’s disease, late-onset diabetes mellitus and other diseases, there is a need for ongoing monitoring of intracellular Zn levels as we age. The priority should be to maintain healthy Zn levels during aging rather than to try and correct Zn deficiency after it has occurred. This is particularly important in the case of neuronal loss, where only limited regeneration is possible. In other situations, therapeutic manipulation of levels of intracellular labile Zn in vivo by supplementation where apoptosis is excessive (e.g., neurodegenerative disorders) or chelation where apoptosis is insufficient (e.g., autoimmunity and malignancy) may provide new therapeutic strategies. The issue will be how best to supplement the relevant intracellular pools in different affected tissues.
Another area that is very much underdeveloped is the cellular biology of Zn, in particular, the fluxes of labile Zn that either participate in or result from biological and pathological processes. There is relatively little knowledge of the differences in content and distribution of pools of Zn in different cells, tissues and organs at different stages of development, in different metabolic states and in local or systemic disease. Nor do we understand how these fluxes of Zn interact with the pathways of apoptosis. Much has yet to be learned about how cells handle and use Zn, concentrate it in organelles and insert it in Zn metalloproteins. How, for instance, do labile Zn-rich pancreatic islet cells differ in the Zn uptake, and how is Zn incorporated from relatively labile Zn-poor cells such as lymphocytes? After exocytosis, where and how is the secretory Zn reacquired? It is only when the cellular biology of labile Zn is better understood will the full implications of Zn-relatedapoptosis become apparent.
Fraker and Telford (1997
) concluded their recent
reappraisal of Zn and apoptosis with the comment, "Finally, the more
important question may be whether so-called inhibitors of apoptosis
actually provide cells with long term protection or only a very
temporal blockage that is subsequently overridden." Perhaps the issue
that needs clarification is the extent to which Zn cooperates with
Bcl-2 and other survival factors. We must be prepared to study the
effect of small physiological Zn fluxes on the background of
up-regulation of other apoptotic suppressors rather than to simply
test the effect of high, potentially toxic concentrations of Zn in
isolation.
|
FOOTNOTES |
|---|
3 Abbreviations used: Zn, zinc; [Zn2+]i, intracellular zinc concentration.
|
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