Introduction

It has been proposed that zinc became especially important inevolution about 2 billion years ago, when the earth’s atmos-phere was becoming oxygen rich and the development of cellular respiration introduced potentially harmful reactiveoxygen species into cells. At this time, cells began to use Zninstead of Fe or Mn in many proteins, especially those com-municating with DNA.1 Zn, a group IIb metal, is critical forthe functional and structural integrity of eukaryotic cells andtissues and is required for events as diverse as gene expres-sion, DNA synthesis, enzymatic catalysis, hormonal storageand release, tissue repair, neurotransmission, memory and thevisual process.2,3 Zn has properties advantageous for a role incytoprotection as it can protect proteins and nucleic acidsfrom oxidation and degradation, while stabilizing the micro-tubular cytoskeleton and cellular membranes.1,2

Zn may have been particularly useful from an evolution-ary viewpoint, not only because it was able to bind to cys-teine, histidine and glutamate amino acids in proteins andthereby create protein folds associated with novel functions(as in Zn fingers), but because it exists in only one oxidationstate (II) and therefore cannot undergo redox reactions com-monly responsible for the generation of potentially damagingoxy-radicals. In fact, by binding to thiol groups in proteins,

Zn can reversibly protect them from oxidation.1 These anti-oxidant properties of Zn were probably important in thecontext of regulation of apoptosis, which was also emergingat this time point, because cells were becoming damaged byoxidative stress. From this viewpoint it is interesting that Znshares features in common with the anti-apoptotic proteinBcl-2, which emerged later in evolution and which also hasanti-oxidant properties.4 Both Zn-deficient rodents2,5–7 andBcl-2 knockout mice4 are growth retarded, have severeimmunodeficiency associated with massive thymic atrophyand depletion of CD4+ T cells and exhibit greying of the fur.

Suppression of premature apoptosis may be, therefore, acritical physiological function of Zn. For detailed reviews onthe earlier papers relating Zn and apoptosis, the reader isreferred to Zalewski and Forbes,6 Fraker and Telford7 andZalewski.8 In this paper, we will try to relate Zn to currentconcepts of the regulation of apoptosis, and attempt to iden-tify some of the questions that need addressing.

Cellular biology of zinc and zinc fluxes

Our current understanding of the cellular biology of Zn has its origins four decades ago with the identification of stoichiometric quantities of Zn in metalloenzymes and theuse of colorimetric stains for tissue Zn (e.g. dithizone). Sincethen, Zn has been shown to be a structural and/or functionalcomponent of more than 200 metalloenzymes, as well asnumerous Zn finger transcription factors. In addition tohousekeeping metabolic roles of Zn, this metal also playscritical roles in many physiological processes.2 The diversefunctions of Zn extend from tightly bound pools within Zn

Immunology and Cell Biology (1999) 77, 272–278

Curtin Conference

Regulation of caspase activation and apoptosis by cellular zincfluxes and zinc deprivation: A review

FUGUI CHAI, AI Q TRUONG-TRAN, LIEN H HO and PETER D ZALEWSKI

Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia,Australia

Summary Non-toxic agents that target intracellular signalling pathways in apoptosis may have potential thera-peutic use in many diseases. One such agent is the transition metal Zn, a dietary cytoprotectant and anti-oxidant,which stimulates cell proliferation and suppresses apoptosis. Zn is maintained in discrete subcellular pools that arecritical for the functional and structural integrity of cells. The present review initially describes the current state ofknowledge on the cellular biology of Zn, especially the critical free or loosely bound (labile) pools of Zn, whichare thought to regulate apoptosis. We then review the evidence relating Zn to apoptosis, including studies from ourlaboratory showing potent synergy between intracellular Zn deficiency and the short chain fatty acid butyrate ininduction of caspase activation and the downstream events of apoptosis. Our studies have also reported the suppressive effects of micromolar concentrations of Zn on caspase-3 activation in cell-free models. Other key issuesthat will be discussed include the identification of the putative molecular targets of Zn and the evidence that sys-temic changes in labile Zn levels are sufficient to alter susceptibility to apoptosis and lead to physiopathologicalchanges in the human body. Finally, we propose that labile Zn may serve as a coordinate regulator of mitosis andapoptosis to regulate tissue growth.

Key words: apoptosis, caspase, mitosis, zinc, Zinquin.

Correspondence: Dr P Zalewski, Department of Medicine, Uni-versity of Adelaide, The Queen Elizabeth Hospital, Woodville, SA5011, Australia. Email: <pzalewski@medicine.adelaide.edu.au>

Received 2 February 1999; accepted 2 February 1999.

APOPTOSIS FEATURE OA 825 EN

proteins, which constitute a largely fixed pool of cellular Zn(associated with metalloenzymes and zinc finger proteins), tomore dynamic, labile Zn pools, which are loosely associatedwith proteins, lipids, cytoskeletal processes or sequestered invesicles. These labile pools, which we believe are importantin cytoprotection and the regulation of apoptosis, amongother things, are readily influenced by Zn deprivation or sup-plementation.2,6,8–11

There are no stores of Zn in the body and Zn homeostasisis regulated largely by intestinal absorption and excretion. Zn,either ionic or bound to ligands, such as histidine, is absorbedin the distal duodenum and proximal jejunum.3 Uptake isprobably largely via the recently cloned divalent cation trans-porter (DCT)-1, which also transports a number of other diva-lent metal ions, including Fe. The 561 amino acid DCT-1protein, with 12 putative membrane-spanning domains, transports metal ions by an active mechanism that is protoncoupled and dependent on the cell membrane potential.12

Following uptake across the enterocyte membrane, Zn can beused locally for nutritional purposes or shuttled through thecell. The latter is mediated by the 77 amino acid cysteine-richintestinal protein (CRIP), a diffusible intracellular carrier intranscellular mucosal Zn transport. Cysteine-rich intestinalprotein contains two cysteine- and/or histidine-rich Zn fingermotifs, which bind Zn and may participate in protein–proteininteractions.13,14 Excess Zn associates with another cysteine-rich protein, metallothionein, and is trapped in the mucosa,thereby limiting absorption and maintaining Zn homeostasisduring periods of high Zn intake.14 At the serosal end, Znefflux is mediated by ZnT-1, a protein with six putative mem-brane-spanning domains and a large cytoplasmically locatedhistidine-rich loop between domains 4 and 5 that may serve tobind cytosolic Zn.14 Following efflux, Zn is carried in theportal blood supply to the liver and other tissues, largely as acomplex with albumin. The rapidity of uptake of 65Zn into the

nuclei of liver, kidney and spleen cells, shortly after ingestion,argues for a direct pathway of exchangeable Zn from dietaryto intracellular labile pools.15

Much less is known about Zn uptake across membranes ofother cells in the body and its incorporation into proteins andorganelles. The transporter involved in cell uptake has notbeen identified, but ZnT-1 is thought to play a major role inZn efflux during times of Zn overload.16 Excess Zn may alsobe complexed with metallothionein. The introduction of sen-sitive, membrane-permeable UV-excitable Zn fluorophores,such as TS-Q and Zinquin, has revealed discrete subcellularpools of Zn in intact spermatozoa, pancreatic islet cells,neurons and other cells.9–11,17 In particular, the identificationof intravesicular pools of Zn (or zincosomes) in Zn-supple-mented cells led to the cloning of a second specific Zn trans-porter, ZnT-2, which is closely related to ZnT-1 and isassociated with vesicular membranes (Fig. 1).16 Also likely tobe associated with zincosomes are the cytoskeletal proteinactin and the signal transduction enzyme protein kinase C(PKC), which form phospholipid- and Zn-dependent com-plexes in Zn-loaded cells.18 We are currently using doublelabelling techniques to look for localization of these mole-cules, as well as various apoptosis-related regulatory proteins(e.g. caspases), around zincosomes.

Intracellular labile zinc and apoptosis

Historically, the first evidence that Zn may be important inthe regulation of apoptosis came from a study in 1977 byElmes,19 who reported greatly increased frequencies of apop-totic cells in the small intestinal crypts of zinc-deficient rats.She proposed that this apoptosis was triggered by a failure ofDNA synthesis in these cells. Subsequent studies by the sameauthor and by others showed that the frequency of apoptoticcells was also markedly increased in many tissues of adult

Zinc-regulated caspase activation 273

Figure 1 Model of a zincosome. The membrane is spanned by the Zn transporter molecule ZnT-2, which contains six putative mem-brane domains and a large histidine-rich loop, thought to be a Zn-binding site, at the cytosolic end. ZnT-2 sequesters Zn in the vesicle.Other proteins associated with the vesicle include actin and protein kinase C (PKC). The evidence that caspase-6, or other caspases, arelocalized to zincosomes remains speculative.

animals, as well as in the neuroepithelium in foetal rats borneby Zn-deficient dams.6 The extent to which apoptosis occursin Zn-deficient humans is less clear, due to the unavailabilityof tissues and organs. Increased apoptosis in Zn-deficienthumans has been inferred from studies of peripheral leuko-cytes of patients with Down syndrome20 and may alsoaccount for the characteristic vesicular skin lesions andsevere T cell depletion in Zn malabsorption syndromes suchas acrodermatitis enteropathica.3

Increased apoptosis in vivo could either be a direct con-sequence of a decrease in intracellular Zn in the affectedcells or secondary to changes in other tissues. There is atleast one example of the latter, where apoptosis in thethymus of Zn-deficient rodents was at least in part due toexcessive levels of circulating glucocorticoids triggered by aZn deficiency-associated stress response.7 This appears to bean exception, however, and evidence from in vitro studiesindicates that apoptosis can result directly from a decline inintracellular Zn induced by culture of cells in either Zn-freemedium or in the presence of membrane-permeant Zn chelators, such as N,N,N’,N’-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN).6,10

Not only can Zn deficiency directly induce apoptosis, butthere is also a potent synergy between Zn depletion and sub-optimal concentrations of other apoptotic inducers. Thus, invitro, TPEN potentiated apoptosis of colchicine-treated lym-phoid cells10 and HeLa cells overexpressing HIV-1 Tatprotein.21 In vivo, colchicine was more toxic to intestinalepithelial cells in Zn-deficient rats than in normal rats.22 Alowering of intracellular labile Zn appears therefore torender cells more susceptible to cell death-inducing agents.In contrast, increasing intracellular Zn by supplementationprotects various types of cells (e.g. anterior and stromal keratinocytes in rabbits23 and hippocampal neurons ingerbils24) from apoptotic death in vivo, while supplementa-tion in vitro prevents apoptotic DNA fragmentation and, atleast in some cases, cell death in diverse types of cellstreated with various apoptosis inducers.6 These studies showthat Zn levels in cells can be manipulated sufficiently toaffect susceptibility to toxins. Studies with mouse thymo-cytes and human chronic lymphocytic leukaemia cells

revealed a strong inverse correlation between the level ofintracellular labile Zn and the extent of apoptotic DNA frag-mentation, such that relatively small changes in labile Znwere able to cause large changes in the susceptibility of cellsto apoptosis, and suggested that a threshold concentration inintracellular Zn may exist, below which apoptosis isinduced.10

Effects of priming cells with butyrate on zinc chelator-induced activation of caspase-3-like enzyme

To further investigate the mechanism of action of Zn in apop-tosis, we have been using a two-stage model of apoptosis inJurkat T lymphocytic leukaemia cells and other cancer celllines. In this model,25 priming the cells for 18 h with a sub-optimal concentration (1 mmol/L) of the short-chain fattyacid butyrate renders the cells highly sensitive to induction of caspase activation and apoptosis by a second signal (e.g.staurosporine), added for the final 2 h. Priming with butyratealso renders the cytosol highly sensitive to activation ofcaspase-3 by the addition of cytochrome c, a cofactor for theproteolytic processing of caspase-3 precursor. Butyrate is ahistone deacetylase inhibitor that causes hyperacetylation ofhistones, leading to chromatin relaxation and enhancedaccessibility of gene regulatory elements to transcriptionfactors. We propose that in stage one (priming), there is up-regulation of a protein(s) that primes the cytosol for caspase-3 activation by agents (such as staurosporine) that releasecytochrome c from mitochondria (stage 2).

The Zn chelator TPEN is also a potent inducer of stage 2;addition of TPEN for 2–3 h, after priming cells with butyrate,results in a dramatic enhancement of caspase-3 and caspase-6activity, as determined by fluorogenic substrate assays. Theseeffects of TPEN are prevented by loading the cells with Znusing the Zn ionophore pyrithione, suggesting that they aredue to chelation of Zn rather than some other metal (Truong-Tran et al., unpubl. data, 1998). Furthermore, Zn loadingduring stage 2 prevented caspase-3 activation by stau-rosporine or by the addition of cytochrome c to the cytosol(Truong-Tran et al. unpubl. data, 1998). These findings aresummarized in Fig. 2.

F Chai et al.274

Figure 2 Schema of a two-stage model: butyrate priming and Zn chelator execution. When cancer cell lines are treated with low con-centrations (e.g. 1 mmol/L) of butyrate for 18 h, they become highly susceptible to induction of apoptosis by the Zn chelator N,N,N’,N’-tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN). Within 2–3 h of TPEN addition, caspase-3 is activated, leading to cleavage of thesubstrates poly(ADP-ribose) polymerase (PARP), p21Waf1 and caspase-activated endonuclease (CAD). Extracts of butyrate-primed cellsalso show enhanced sensitivity to cytochrome c (cyt-c)-induced caspase-3 activation and this is blocked by exogenous Zn. Events involvedin priming by butyrate include histone hyperacetylation and essential new protein synthesis.

Zinc-regulated caspase activation 275

Putative biochemical targets of zinc in apoptosis

The precise step in the cascade of signalling events that is sen-sitive to Zn remains unclear. In 1992, Cohen and colleagues26

observed that Zn supplementation of dexamethasone-treatedthymocytes in vitro blocked the transition from peripheralnuclear chromatin condensation to nuclear collapse. Theyattributed this to specific inhibition of the Ca/Mg-dependentendonuclease that cuts linker regions between nucleosomes ina Ca- and Mg-dependent manner, leading to DNA fragmenta-tion, as seen by ladder patterns on electrophoresis gels.Earlier studies identified this endonuclease as a sensitivetarget of Zn.27 At a relatively low concentration (50 µmol/L),Zn strongly inhibited Ca-induced DNA fragmentation in isolated nuclei.27

Like Bcl-2, Zn may have multiple intracellular targets inthe apoptotic signalling cascade. The first evidence that Znmay affect intracellular targets in apoptosis other than theCa/Mg-dependent endonuclease came from studies by Lazeb-nik and colleagues, who showed that a more sensitive targetof Zn was present in the cytoplasm.28 In these studies, theyshowed that cytosol from cells primed to apoptose inducedchromatin condensation and DNA fragmentation in isolatedhealthy nuclei and that Zn suppressed these effects, evenwhen added only to the cytoplasm. Subsequent studies iden-tified the active component of the cytoplasm as an aspartate-specific protease CPP-32 (renamed caspase-3).29 Zn did notblock the activity of caspase-3 to cleave its cellular substrates,but rather blocked the mechanism by which caspase-3 is activated from the inactive zymogen precursor.30 Unpublishedstudies from our laboratory have confirmed this finding andsuggest that Zn supplementation of cells suppresses a stepprior to, or during, the activation of caspase-3. Using a cell-free system, in which addition of cytochrome c to the cytosolof healthy cells triggers the proteolytic conversion of pro-caspase-3 to the active enzyme, we observed that addition ofZn at the same time as cytochrome c blocked activation ofcaspase-3 by 50%; there was no effect of Zn when added aftercaspase-3 activation (Truong-Tran et al., unpubl. data, 1998).

A similar finding using western blotting to track caspase-3processing in HL60 cells has recently been reported.31

One possible target of Zn is Mch-2α/caspase-6, which isknown to cleave and activate the proenzyme form of caspase-332 and to be highly sensitive to Zn,30,33 being suppressed atmicromolar concentrations.33 There is as yet no physico-chemical explanation for the selective effects of Zn on thiscaspase and it still remains to be determined whethercaspase-6 and the cleavage of its major cellular substrates,the nuclear lamins, are influenced by intracellular concentra-tions of Zn.

Another possible target is caspase-9, or associated proteins,which are known to mediate the cytochrome c-dependent activation of precursor caspase-3 processing29 (Fig. 3). Activa-tion of caspase-9, in the presence of the adaptor moleculeApaf-1 and the cofactors cytochrome c and dATP, results in theprocessing of precursor caspase-3 to small and large subunitsthat reassemble to form the active caspase; this then cleavesvarious substrates including poly(ADP-ribose) polymerase(PARP), p21Waf1 and the caspase-activated endonuclease(CAD). Caspase-3 processing is inhibitable by Bcl-X

Land

other Bcl-2 family proteins. We propose that it is also sup-pressed by Zn (Fig. 3).

Another recent study has implicated Zn in the regulation ofthe anti-apoptotic Bcl-2-like and pro-apoptotic Bax-like mito-chondrial membrane proteins.34 Fukamachi and colleaguesreported a significant increase in the ratio of Bcl-2/Bax in Zn-supplemented U937 cells.34 This ratio is thought to determinethe susceptibility of damaged cells to apoptotic death, forinstance, high ratios are thought to increase cellular resistanceto apoptosis.4 Other potential targets for Zn are the micro-tubular cytoskeleton, which is stabilized by Zn and disruptedin apoptosis,6 and the cytoplasmic glucocorticoid receptor inthymocytes.7 At least in neurons, Zn may also influence eventsat the level of gene expression, because Zn deprivation-induced apoptosis is suppressed by cycloheximide.35

A number of questions still remain to be answered. First,it is not clear whether the targets of Zn identified by Zn supplementation experiments are indeed physiological targets.

Figure 3 Model for Zn-regu-lated caspase-3 processing. Theprocessing and activation of theproenzyme form of caspase-3 iscaspase mediated. Both caspase-9and caspase-6 have been impli-cated in processing. Caspase-9-mediated activation requires anadaptor protein, Apaf-1, and co-factors dATP and cytochrome c.The process is inhibited by Bcl-2family members (e.g. Bcl-X

L) and

by Zn.

In particular, very high (often millimolar) concentrations ofZn were added to cells or cell extracts in these studies. Tosome extent, these high concentrations were compensated forby the presence of Zn-chelating substances (e.g. EGTA,EDTA and dithiothreitol) in the buffers. For example, we havefound that the addition of millimolar concentrations of ZnSO

4

to the buffers used in the cell-free assay of apoptosis, resultsin free concentrations of Zn that are only in the micromolarrange, as determined using a Zinquin fluorescence assay(Truong-Tran et al., unpubl. data, 1998). Certainly, activity ofcaspase-6 and mechanisms involved in the activation ofcaspase-3 are sensitive to more physiological (micromolar)concentrations of Zn.

A second area of concern is that most of the conclusionsrelating to possible cellular targets of Zn have been basedsolely on Zn supplementation studies. Because the most com-pelling evidence for a physiological role for Zn in regulationof apoptosis comes from Zn deficiency studies in vivo, it isessential that future studies also address the intracellulartargets that are affected by a lowering of intracellular Zn.Whether the same targets are affected by both Zn deprivationand Zn supplementation remains unclear.

Further clues to the identity of the key targets of Zn maycome from a better understanding of the functions of discretesubcellular pools of Zn. Potential targets of Zn may exist inthe nucleus (e.g. Ca/Mg endonuclease), cytosol (e.g. caspase-6), microtubules or mitochondria (e.g. Bcl-2/Bax). Multiplesubcellular pools are probable, because Zn inhibits apoptosisregardless of whether added to isolated nuclei27 or tocytosol.28 In Zn-supplemented cells, there is intense labellingof membrane-enclosed vesicles.10 Future studies from ourlaboratory will investigate the interaction between thesepools of Zn and caspase-6, Bcl-2 and other apoptotic regula-tors, using double labelling and cell fractionation techniques.

Physiopathological implications

There remains a large gap in our understanding betweenintracellular Zn regulation and its consequences for bothphysiological processes in the body and their pathologicalabnormalities in disease. Of particular interest is whetherfluxes of tissue and organ Zn concentrations during physio-pathological events influence the susceptibility of cells toundergo caspase activation and apoptosis. Both conditions inwhich there are primary or secondary changes in total bodyZn status and conditions in which the body Zn levels may notchange but rather undergo redistribution or temporary fluxesare to be considered here.

Tissue Zn deficiency may be precipitated not only by malnutrition or malabsorption, but may also occur secondaryto ageing, obesity and in diseases such as diabetes mellitus,where there are excessive Zn losses in urine.3 Whether thesesecondary changes in Zn homeostasis are of sufficient mag-nitude to influence cell susceptibility to apoptosis is notknown and needs further study. One approach would be tostudy the susceptibility of neutrophils to apoptosis in vivo,because it is known that neutrophil Zn levels decline in evenmildly Zn-deficient humans in ageing.36 Peripheral leuco-cytes of patients with Down syndrome, a disease associatedwith Zn deficiency, have been reported to have an increasedincidence of features typical of an early stage of apoptosis

and this was abolished following long-term oral Znsupplementation.20 Decreased uptake and utilization of Zn byaged cells36 could contribute to the increased susceptibility toapoptosis of senescent cells, as occurs in patients withAlzheimer’s disease. Tissue Zn deficiency in diabetes melli-tus3 may be a factor in the depressed immune response inthese patients by increasing apoptosis of T cells. Lowered Znlevels may also directly affect the diabetic state by renderingthe insulin-producing pancreatic β-cells more susceptible toapoptosis by autoimmune cytotoxic T cells or by chemicaldiabetogens. However, increased levels of Zn or decreased Zndependence in some tumour cells may be a factor in their relative resistance to apoptosis and accelerated growth.37

At this stage, we do not know whether all cells in thebody are rendered more susceptible to apoptosis in Zn defi-ciency or whether only some types of cell are more sensitive.Zn deficiency primarily increases apoptosis in those tissuesundergoing rapid cell turnover (e.g. thymus, epidermis, testisand intestinal crypts).6 Is it because the cells in these tissuesare primed for apoptosis or because cycling cells are prefer-entially sensitive to Zn depletion? The relationship betweenlevels of labile intracellular Zn and susceptibility to apopto-sis needs clarification. Most tissues contain approximately20–200 µg/g zinc; however, the retina, prostate, pancreaticislets and sperm contain much higher amounts(800–3000 µg/g).2,9 Of particular interest are pancreatic isletcells, which are extremely rich in labile Zn11 and also veryresistant to apoptosis38 relative to the surrounding acinarcells. Because the labile Zn is largely in the secretorygranule pool in islet cells, this raises the question of whethersecretory Zn can also influence apoptosis, either of neigh-bouring cells or at an intracellular level.

Which of the classical features of Zn deficiency arecaused by an increase in apoptosis? One could make a casefor the T cell-related immunodeficiency, the general wastingof tissues and the different types of skin lesions. Mori andcolleagues39 have proposed a role for increased apoptosis inthe formation of vesicular skin lesions in patients withacquired zinc deficiency, although not in the formation ofhyperkeratotic skin lesions.

Zn fluxes have now been shown to accompany a numberof physiological events in the body, including secretion,mitosis, spermatogenesis, inflammation, tissue repair andneurotransmission.2,6,8–11,17 The implications of these fluxesfor regulation of apoptosis have yet to be determined. Thefluxes of Zn into the liver during inflammation and intosperm heads during ejaculation are likely to be involved incytoprotection. Fluxes of Zn during the G

1phase of mitosis,

in contrast, may play a pivotal role in both cell proliferationand cell death. Because Zn both promotes mitosis37 and sup-presses apoptosis, it may serve to coordinate these twoopposing growth regulatory processes. Hence, the enhance-ment of mitosis and suppression of apoptosis in Zn-repletetissues would favour tissue regeneration, while tissue involu-tion and atrophy would result from the simultaneous sup-pression of mitosis and enhancement of apoptosis in Zndeficiency. Cellular Zn uptake is enhanced by growth factors,hormones and other agents that promote mitosis and suppressapoptosis.6,37 Because cells need both growth factors and Znto pass the G

1/S restriction point (a cell cycle decision point

about 2–3 h prior to onset of DNA synthesis), after which

F Chai et al.276

neither is required,37 events close to this restriction point maybe critical to the mechanism by which cell birth and death arecoordinated.

Conclusions

This review has attempted to address the following threehypotheses: (i) that a specific pool(s) of intracellular labileZn regulates apoptosis; (ii) that systemic changes in Zn levelsin the body, due to dietary factors, altered physiological statesor disease, can sufficiently alter labile intracellular Zn so asto change cell susceptibility to apoptosis; and (iii) that thisaltered susceptibility to apoptosis contributes to physio-pathological changes in the body. The first hypothesis is nowwell supported by the numerous studies of the effects of Zndeprivation or supplementation, on either the spontaneousrate of apoptosis or the susceptibility of cells to induction ofapoptosis by other agents, as well as by studies on the effectsof Zn on various components of the apoptotic signallingpathway. That Zn is a physiological regulator of apoptosis,however, still needs to be proved. The best evidence comesfrom Zn deficiency studies in experimental animals, since thein vitro studies are still fraught with problems in distinguish-ing the physiological effects from the pharmacological andtoxicological effects of Zn. Most published studies have usedexcessively high amounts of Zn to supplement cells orextracts, thereby creating supraphysiological concentrations.A second reservation concerning many of these studies is thatend-points measured often did not include morphology andcell viability. It is now clear that caspase activation and DNAfragmentation can be suppressed by Zn without necessarilypreventing cell death.7

We have begun to identify the intracellular pool(s) of Znthat mediate suppression of apoptosis, in the hope that thiswill provide new clues to the biochemical targets of Zn. Asimilar strategy with Bcl-2 led to its identification as an outermitochondrial membrane protein and the discovery of its rolein regulating cytochrome c release and subsequent activationof caspases. Co-localization studies using Zinquin and spe-cific antibodies may indicate novel interactions between Znand other components of the cascade. Of the four caspasestested to date, caspase-6 is the only one to be affected byphysiological concentrations of Zn. Whether caspase-6 isunique in this respect will only be known when the remain-ing 10 or more caspases are tested. From a cursory glance atthe primary structure of caspase-6, there is no obvious reasonfor its Zn sensitivity; Zn-binding studies coupled with site-directed mutagenesis should indicate the region(s) of themolecule that interact with Zn, while effects of Zn chelatorsmay indicate whether the enzyme is normally saturated withZn in living cells. The use of cell-free models of caspase pro-cessing and apoptosis, in which free Zn concentrations can beset, should be instructive.

From a therapeutic viewpoint, Zn may be administered toprevent or ameliorate degenerative disorders associated witha high rate of apoptotic cell death, like Alzheimer’s demen-tia. With this in mind, there is a need for regular monitoringof intracellular Zn levels during ageing and in diseases asso-ciated with acquired Zn deficiency. The development ofbetter tests for intracellular Zn deficiency is urgentlyrequired. Recent developments in the introduction of Zn

fluorophores and stable Zn isotopes for in vivo studies as wellas the cloning of the first mammalian Zn transporter mole-cules should stimulate research into the cellular biology of Znand the fluxes of Zn in physiopathological processes. One ofthe challenges of current apoptosis research is to understandnot only how the different protein regulators of apoptosiswork in concert to regulate cell death, but also how these pro-teins cooperate with metals like Zn. This will have implica-tions for understanding why many growth factors are Zndependent and why changes in intracellular levels of Zn haveopposite effects on the two major processes which controltissue growth, proliferation and apoptosis.

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