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Apoptosis2003;8: 6170

C 2003 Kluwer Academic Publishers

On the role of Hsp27 in regulating apoptosis

C. G. Concannon, A. M. Gorman and A. Samali

Department of Biochemistry and National Centre for Biomedical Engineering Science,National University of Ireland, Galway, Ireland

Heat shock proteins (Hsps) comprise several differentfamilies of proteinsthat are induced in response to a widevariety of physiological and environmental insults. Onesuch protein which is highly induced during the stressresponse is a 27-kDa protein, termed Hsp27 whose ex-pression is seen to correlate with increased survival in re-

sponse to cytotoxic stimuli. It has been shown to preventcell death by a wide variety of agents that cause apop-tosis. Hsp27 is a molecular chaperone with an ability tointeract with a large number of proteins. Recent evidencehas shown that Hsp27 regulates apoptosis through anability to interact with key components of the apoptoticsignalling pathway, in particular, those involved in cas-pase activation and apoptosis. This article will review re-cent advances in the field and will address some of thepotential mechanisms by which Hsp27 functions as ananti-apoptotic molecule.

Keywords:apoptosis; caspase; Hsp27; stress.

During the course of evolution, cells have developedcomplex dynamic mechanisms to respond to the manyphysiological and environmental insults they encounter.Analysis of these responses has led to the discovery ofhighly conserved proteins, termed heat shock proteins(Hsps), whose synthesis is transiently induced in responseto low levels of stress, in a process referred to as the stressresponse.1 Hsps encompass several groups of proteins andmay be divided into five major families on the basis oftheir size, structure and function:2 the Hsp110, Hsp90,Hsp70, Hsp60 and small Hsp families. Hsps were origi-

nally named because of their rapid inductionin response toelevated temperatures,3 however, it has since been shownthat a wide variety of different physical, chemical and bi-ological stimuli are also capable of inducing Hsps includ-ing oxidative stress, heavy metals, amino acid analogues,osmotic stress, and metabolic poisons.2

Some Hsps are constitutively expressed and increase inresponse to stress while the expression of others is only

Correspondence to: A. Samali, Department of Biochemistry andNational Centre for Biomedical Engineering Science, NationalUniversity of Ireland, Galway, Ireland. Tel: +353-91-750393;

Fax: +353-91-512504; e-mail: [email protected]

induced following exposure of cells to environmental andphysiological stresses. In unstressed cells, constitutivelyexpressed Hsps are essential for maintaining cell home-ostasis, functioning as molecular chaperones to facilitatethe transport, folding and assembly of polypeptides. Dur-

ing the stress response the intracellular levels of manyHsps rapidly increase due to the increased concentrationof unfolded proteins that occurs. The induced expressionof these Hsps is seen to be cytoprotective, protecting cellsfrom toxic insult and preventing their demise.4 Such in-ducible Hsps bind to damaged and misfolded polypep-tides and mediate their refolding or degradation, thusprotecting cells from potential deleterious effects and pro-moting cell recovery.5 This review will focus on Hsp27,which is one of the main inducible Hsps and has recentlybeen reported to be a molecular inhibitor of apoptosis, aproperty which contributes to its cytoprotective effects.

Hsp27 and the small Hsp family

Hsp27 belongs to a family of abundant and ubiquitousstress proteins, the small heat shock proteins (sHsps),which are detectable in virtually all organisms fromprokaryotes to mammals.6 sHsps vary in size from 15 to30 kDa and to date nine different members of this familyhavebeen identified:Hsp27, p20,HspB3, MKBP/HspB2,HspB8, HspB9, cvHsp, -A crystallin and -Bcrystallin.714 Although members of this family share lowamino acid homology, they are grouped together based

on similar structural and functional properties, with allsHsps having a conserved core region that was first identi-fied within the crystallin proteins of the vertebrate eye.15

This domain, termed the crystallin box, comprises 80100 amino acids in the C-terminus of the protein andhas an IgG-like fold, which is followed by a short morefreely flexing C-terminal extension. In contrast, the N-terminus of sHsps is much more variable both in sequenceand length16 and contains the WDPF motif, which is in-volved in oligomerization of the protein.17

Within unstressed cells Hsp27 levels are generally lowandit exists predominantly as a large oligomeric unit of up

to 800kDa, usually comprisedof sixtetrameric complexes

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of the protein. The size of this oligomeric unit is depen-dent on a number of physical and chemical parameters,which include temperature,pH, ionic strength andthe de-gree of phosphorylation of the individual monomers. Dur-ing the stress response an increase in the level of Hsp27expression is preceded by a phosphorylation-induced reor-ganization of the multimeric status of the protein. Phos-phorylation occurs on three different serine residues, Ser-15, Ser-78 and Ser-82; resulting in the redistributionof the large oligomer into smaller tetrameric units.18,19

This induced phosphorylation of Hsp27 is catalysed byMAPKAP kinases 2 and 3,2022 which in turn are ac-tivated through phosphorylation by p38 MAP kinase.23

Recent evidence suggests that Hsp27 may also be phos-phorylated by the delta isoform of protein kinase C, 24

however, this appears only to occur in a stimulus-dependent manner,25 such as treatment with phorbol es-

ters. The increasedphosphorylation of Hsp27 is detectableseveral minutes after exposure to stress, followed by asubsequent increase in the expression levels of the pro-tein within several hours.26 In general, the induction ofHsp27 is transient and the protein returns to basal lev-els after removal of the stress event. Interestingly though,increased expression of Hsp27 is transiently induced atspecific stages during development and cell differentia-tion and occurs concomitantly with the differentiation-mediated decrease of cellular proliferation.27 Indeed, ex-periments suggest that aberration of this accumulationin mouse embryonic stem cells is sufficient to abort the

differentiation process leading to the death of the cells.

28

Hsps as emerging anti-apoptoticmolecules

It has been known for some time that cells induced toaccumulate Hsps subsequently become more tolerant tocytotoxic insults, a phenomenon termed thermo-tolerance.1,29,30 This increased cellular survival can becorrelated with the expression of Hsps and their ability toprevent cell death.

Within tissues cell death can occur via either necro-

sis or apoptosis. Necrosis is a passive form of cell death,whichoccurs mainly underpathologicalconditions, wherea rapid loss of ion-flux control leads to the swelling andrupture of the cell and its organelles. In contrast, apop-tosis is a controlled, energy-dependent form of cell deathinvolving the use of signal transduction pathways in bothits initiation and execution. Characteristic hallmarks ofapoptosis include condensation of nuclear chromatin, cy-toplasmic shrinkage, membrane blebbing, nuclear frag-mentation and formation of apoptotic bodies. Inductionof apoptosis can occur by means of a large variety of stim-uli including cytokines, cytotoxic drugs, oxidative stress

and ionising radiation (for review see ref. 31).

Diverse stimuli trigger apoptosis by activating one ormore signal transduction pathways, which then convergeto activate a conserved family of aspartic-acid specificcysteine proteases, referred to as caspases. Caspases areconstitutively expressed within cells as inactive precursorzymogens and upon initiation of apoptosis undergo spe-cific proteolytic cleavage, resulting in their subsequentactivation.32 Once activatedthey orchestrate thedemise ofthe cell through the cleavage of a specific subset of cellularsubstrates,33 resulting in the characteristic biochemicaland morphological changes associated with apoptosis.34

Activation of apoptosis proceeds either via an intrin-sic or extrinsic signal transduction pathway. The extrin-sic pathway involves the binding of death ligands to cellsurface receptors (e.g., Fas/CD95/Apo-1) resulting in therecruitment of the adaptor molecule FADD to the cy-tosolic end of the receptor leading to the formation of

a death receptor complex at the plasma membrane andthe resultant activation of pro-caspase-8 and thereby pro-caspase-3.35 On the other hand, the intrinsic pathway isinitiated through the release of cytochrome c from theintermembrane space of mitochondria.36 Cytochrome ccrosses the outer mitochondrial membrane to the cytosolwhere it acts as a co-factor for Apoptosis Protease Activat-ing Factor 1 (Apaf-1) function in the presence of dATP.The binding of cytochromec/dATP to Apaf-1 promotesits oligomerization and the recruitment of pro-caspase-9.37 The formation of this caspase-activating complex,termed the apoptosome, results in the activation of pro-

caspase-9, which further amplifies the caspase cascade byits ability to process its own pro-enzyme as well as effectorcaspases including pro-caspase-3.38

Recent reports have pointed to the mitochondrion as acentral control point for the integration of death signalsduring apoptosis. Many of the key molecules involved inapoptosis are located within or attached to the mitochon-drion. These molecules include cytochrome c,39 AIF,40

Smac/Diablo41 and pro-caspase-3,42 and when releasedinto the cytosol function both in the initiation and execu-tion of the apoptotic program. It is therefore unsurprisingto find that the mitochondrion serves as a control point forcross-talk between the intrinsic and extrinsic pathways of

apoptosis. In this respect it is well established that the ac-tivation of pro-caspase-8 by death receptors can result inthe cleavage of an endogenous cellular protein, Bid, gen-erating a pro-apoptotic fragment that translocates to themitochondrionwhere it induces cytochrome crelease.43,44

This release of cytochromecfurther amplifies the caspasecascade and is found to be critical in certain cell typeswhere the cross-linking of death receptors alone is not suf-ficient to induce caspase activation without cytochromecrelease. Further reports also suggest that stimuli thatinduce DNA damage and subsequently apoptosis, do sovia translocation from the nucleus to the mitochondrion

of molecules that stimulate the release of cytochromec.45

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Naturally, since the mitochondrion has such a key roleduring apoptosis, many of the endogenous cellular pro-teins that function as crucial determinants of cell deathbring about their anti-apoptotic abilities by acting on mi-tochondria, thereby helping to prevent release of crucialpro-apoptotic proteins.

To datethe best-characterisedendogenous protein mod-ulators of apoptosis are the members of the Bcl-2 familyof proteins.46 Both pro- and anti-apoptotic members ofthis family have been identified and studied within mam-malian cells, where they are seen to function as key deter-minants of cell fate. However, the recent correlation be-tween the expression of Hsps and increased cell survival,has pointed to Hsps as playing a critical role in the regu-lation of the apoptotic cell machinery. Experiments havedemonstrated that Hsp72 or Hsp27 increase cell survivalin response to apoptotic stimuli.4749 It is therefore appar-

ent that during thermotolerance the protective effect ofHsps is due, at least in part, to the ability of these proteinsto inhibit apoptosis.48 Because of their potential role askey death determinants within cells, much attention hasfocused on elucidation of the molecular mechanism bywhich Hsps mediate their anti-apoptotic abilities. Hsp72has been reported to inhibit apoptosis through a directinteraction with Apaf-1, thereby preventing the dock-ing of pro-caspase-9 and its subsequent activation.50,51

Moreover, Hsp90 negatively regulates caspase activity byinhibiting the cytochrome c-mediated oligomerization ofApaf-1.52 In line with these observations interactions be-

tween Hsp27 and critical components necessary for theactivation of caspases have also been observed and thesewill be discussed in detail in the next section.

Mechanism of cytoprotectionby Hsp27

A variety of different roles for Hsp27 during cellular stresshave been proposed to account for the cytoprotective ef-fects seen with increased expression of this protein. Theseinclude its role as a molecular chaperone, direct interfer-ence with the mechanisms of caspase activation, modula-

tion of oxidative stress and regulation of the cytoskeleton.

Hsp27 as a molecular chaperone

Similar to other members of the Hsp family, Hsp27 func-tions as a molecular chaperone to aid in refolding of non-native proteins. It forms complexes with such proteins,thus preventing their non-specific aggregation and allow-ing them to be subsequently restored to their native struc-turein co-operation withATP-dependent chaperones suchas Hsp70.53,54 In contrast to most Hsps, the chaperone

ability of sHsps occurs in an ATP-independent manner.55

The C-terminal of sHsps is responsible for the molecularchaperone function of this family.56

The intracellular accumulation of misfolded proteinscan trigger the stress response leading to increased Hspexpression.57 If excessive amounts of damaged proteinsare present they can form large aggregates which serveas a signal for the induction of apoptosis.58 In stressedcells containing high levels of damaged proteins, the in-creased expression of Hsp27 facilitates the repair or de-struction of these proteins thus promoting cell recovery.This ability of Hsp27 to aid the recovery of stress-induceddenaturation of proteins may increase the survival rateof cells by limiting the levels of misfolded proteins thatcould ultimately be responsible for triggering of apop-tosis. For example, overexpression of Hsp27 is associatedwith an enhanced rate of recovery from nuclear proteinaggregation.59

Increased expression of Hsps during the temperature-induced stress response, is accompanied by a shut-off ofgeneral protein and mRNA synthesis.60 Cuesta and co-workers61 have demonstrated that Hsp27 itself can func-tion as an inhibitor of cellular protein synthesis. Theyshowed that Hsp27 interactedin vitrowith a cap-bindinginitiation factor known as eIF4G, which is essential fortranslation of most cellular mRNAs. This interaction pre-vented the eIF4G factor from initiating the start oftranslation.61 As the synthesis of proteins under stressconditions could potentially result in their incorrect fold-ing and accumulation of protein aggregates, this inter-

action could be a protective mechanism to further limitthe accumulation of misfolded proteins during stress. In-terestingly, expression of Hsp27 has also been reportedto stimulate recovery of RNA and protein synthesis fol-lowing heat shock62 which may provide the cell with asurvival advantage.

Moreover, the molecular chaperone function of Hsp27is responsible for the regulation of apoptosis through theinteraction of Hsp27 with protein kinase B (Akt). Activa-tion of Akt has been demonstrated to inhibit apoptosis ina variety of systems.6367 During both heat and hydrogenperoxide-induced cellular stress, Akt is activated throughan association with Hsp27.68 This Hsp27-mediated ac-

tivation of AKt is likely to contribute to the increasedresistance to apoptosis observed in cells expressing highlevels of Hsp27.64

Inhibition of caspase activation/activity

In recent years, evidence has accumulated to show thatHsp27 can inhibit apoptosis through a direct inhibitionof caspase activation.69,70 As a molecular chaperone, itis not implausible that Hsp27 could regulate the acti-vation of caspases through an ability to interact with

one or more components of the apoptosome complex.



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Figure 1. Hsp27 can regulate various apoptotic pathways. A mechanism by which apoptosis is initiated is due to changes in the

intracellular redox balance and production of reactive oxygen species. This results in changes in the mitochondria and release of pro-

apoptotic factors. Hsp27 can maintain both the redox homeostasis and mitochondrial stability in the cell. Also Hsp27 can bind to both

cytochromec, after its release from mitochondria, and pro-caspase-3 thus preventing apoptosome formation and events downstream

of mitochondrial damage. Apart from the caspase-dependent apoptosis, Hsp27 is reported to block Daxx-mediated apoptosis. Daxx, a

nuclear protein that translocates to the membrane during Fas-mediated apoptosis, binds at one end to the Fas receptor and at the other

to Ask1, thus mediating a caspase-independent apoptosis. Hsp27 can prevent the translocation of Daxx to membrane and its interaction

with Fas.

To this end, several different studies have provided ev-idence of interactions of Hsp27 with critical componentsof the apoptosome (Figure 1). Hsp27 negatively regulatesthe activation of pro-caspase-9 by an ability to interactwith cytochrome c, thus preventing the correct forma-tion/function of the apoptosome complex.71,72 Further-more, it has also been shown that Hsp27 can inhibitcaspase-3 activity by interacting with the pro-caspase-

3 molecule.72,73 Interestingly, although Pandey et al.73were not able to demonstrate a direct interaction betweencytochromecand Hsp27 they conclude that Hsp27 mayact both upstream/downstream of cytochromecrelease ina stimulus-dependent manner.

Recent evidence has demonstrated a significant pool ofHsp27 is located in the mitochondrial fraction of ther-motolerant Jurkat cells.70 This is reminiscent of anotherendogenous anti-apoptotic protein, Bcl-2. Bcl-2 func-tions to inhibit apoptosis by preventing the release ofcytochrome c from the intermembrane space of mito-chondria.74,75 It is therefore interesting to speculate that

Hsp27 may function in a manner similar to Bcl-2. We

have observed that Hsp27 may protect against apoptoticstimuli by blocking the release of cytochrome c.70 Cellsrendered thermotolerant and subsequently challenged byan apoptoticstimulusdid notdisplaythe loss of mitochon-drial membrane potential (m)and subsequent releaseof cytochrome cseen in non-thermotolerantcells.70 Whena similar experiment was performedusingcells transfectedwith antisense directed against Hsp27, the loss ofm

and release of cytochrome cwere not inhibited,70 thusindicating that in thermotolerant cells Hsp27 is respon-sible for inhibition of these parameters. In contrast, Brueyet al.71 did not observe an effect on cytochrome c releaseby Hsp27. A possible explanation for these conflicting ob-servations may lie in the different model systems used inthese experiments. When Hsp27 is transfected into cellsits expression is usually under control of foreign viral tran-scription promoters and not the transcription factor, HSF-1, as is the case during heat shock. Under stress conditionsHSF-1 not only mediates the induction of Hsp27 but alsoa variety of other proteins. Therefore, the differences seen

in these two reports may in fact be because during heat



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shock, Hsp27 localises to mitochondria, a translocationthat may be dependent on the synthesis of other proteinsinduced through HSF-1 activation.

Interestingly, Paul et al.76 have recently reported thatalthough Hsp27 localises to the mitochondrion, this lo-calisation of Hsp27 was not significant in terms of pre-venting cytochrome c release and that Hsp27-mediatedprevention of cytochromecrelease was due to the abilityof Hsp27 to maintain the actin network integrity (dis-cussed in the next section) and prevent the translocationof pro-apoptotic factors from the actin cytoskeleton tothe mitochondrion where they can cause cytochrome c

release.76

The significance of the mitochondrial pool of Hsp27remains to be fully investigated. The idea that Hsps inserttheir protective roles at the level of the mitochondrionis not an entirely new concept. Previously Polla and co-

workers have suggested that mitochondria are the targetsof the protective effects of Hsps against oxidative stress.77

It has also been shown that a novel set of sHsps in PC12cells are localised to the mitochondria where they functionto protect cells against thermal and oxidative stresses.78

These findings add more weight to the suggestion thatthe primary site for the protective effects of Hsp27 maybe the mitochondrion. Since the mitochondrion has sucha key role in the execution of apoptosis it would seem anappropriate location for Hsp27 and other Hsps to elicittheir anti-apoptotic characteristics.

Hsp27 has also been demonstrated to interact with an-

other component of the apoptotic cell death machinery,pro-caspase-3.72,73 It would seem that this interaction iswith the inactive form of the molecule and it is thereforehighly plausible to infer that the binding of Hsp27 to pro-caspase-3 prevents its activation, possibly by preventinginitiator caspases, such as caspase-9, from gaining accessto the necessary residues whose cleavage is required foractivity of the enzyme. Elucidation of the critical residueson both of these molecules which are responsible for theirinteraction should help in further characterizing the role(if any) this interaction plays in regulation of apopto-sis. Interestingly an interaction with pro-caspase-3 hasalso been demonstrated for another of the sHsps, -B

crystallin.79It seems that Hsp27 is adapted to inhibit apoptosis

induced by a variety of different means since the ex-pression of Hsp27 is also associated with inhibition ofapoptosis initiated by the binding of death ligands tocell surface receptors such as Fas.47 The activation ofFas signalling is not primarily associated with the re-lease of cytochrome c and formation of the apoptosome.In this case receptor binding results in the direct acti-vation of pro-casapse-8 with downstream activation ofpro-caspase-3. Since Hsp27 has been demonstrated to in-teract with pro-caspase-3 it may be that the function of

this interaction is primarily to inhibit apoptosis associ-

ated with the activation of death receptors. However, ithas been shown that Hsp27 can interact with Daxx,80 aprotein that has been implicated as a mediator of Fas-induced apoptosis81 (Figure 1). Daxx-mediated apopto-sis is caspase-independent and involves the recruitmentof the apoptosis signal regulating kinase (Ask1), a MAPkinase kinase that activates JNK. Hsp27 interacts withDaxx and blocks its interaction with both Fas receptor andAsk1.80 Alternatively, in some cell systems the activationof death receptors is also associated with amplification ofthe caspase cascade through caspase-8-mediated release ofcytochrome cfrom the mitochondria. It could be arguedthat Hsp27s inhibition of caspases activated by Fas isa consequence of its ability to prevent the cytochromec-mediated amplification of the caspase cascade. However, itseems much more plausible that this is mediated througha synergistic effect of interacting with both cytochromec

and pro-caspase-3.

Hsp27 prevents stress-induced disruptionof the cytoskeleton

Hsp27 has been recognized as a potent regulator of cy-toskeletal dynamics, in particular, actin microfilaments.Within cells, the cytoskeleton mainly functions in main-taining the shape of thecell andthis function is modulatedby both the spatial arrangement as well as the polymeri-sation dynamics of its different elements.82

Several studies haveshown thatoverexpression of Hsp27increases the stability of F-actin microfilaments duringexposure to such stresses as hyperthermia,83 oxidants84

and cytochalasin D.85 During stress the integrity offil-amentous actin structures is disrupted by the disorderlysevering and aggregation of the filaments, resulting in astate that is potentially damaging to for cell morphology.It is suggested that during heat stress, the association ofHsp27 with F-actin may serve as an adaptive responseto changes within the cellular environment, to stabilisethe structure of the cytoskeleton and prevent its disag-gregation. The exact mechanism by which Hsp27 sta-bilises F-actin is poorly understood. Turkey and murine

Hsp25 (a homologue of human Hsp27) have been demon-strated to inhibit actin polymerisation.8688 This activityis dependent on the degree of Hsp25 phosphorylationand on its structural organization. Non-phosphorylatedHsp25 andHsp27monomers areactive in inhibiting actinpolymerization whilephosphorylated monomersand non-phosphorylated oligomeric formsare inactive.83,86 In con-trast, Previlleet al.89 have found that phosphorylation ofHsp25 is not essential for protection of cells against dis-ruption of the actin cytoskeleton and that the protectionof the actin network is probably a consequence of theredox change mediated by Hsp25 rather than a direct

effect of this stress protein on actin. Irrespective of the



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mechanism by which Hsp27 inhibits actin polymerisa-tion it would seem that the loss of Hsp27s actin-cappingability permits actin polymerisation, thereby potentiallystabilising and remodelling the actin cytoskeleton duringstress.

Actin microfilaments are not the only components ofthe cytoskeleton that have been reported to interact withHsp27. An interaction of Hsp27 and another of the sHsp,-B crystallin, with various intermediate filaments hasbeen reported.90 It is proposed that these interactionsmay manage the connections between filaments in cel-lular networks, an event that may be important for thesurvival of cells. Evidence also suggests that Hsp27 co-localizes with tubulin/microtubules, although the signif-icance of this observation is not fully understood.91 It isarguable that the association of Hsp27 with elements ofthe cytoskeleton promotes increased cellular survival by

insulating this dynamic network and preventing its de-struction, which could ultimately result in the death ofthe cell.

Modulation of intracellular redox potential

The generation of high levels of intracellular reactive oxy-gen species (ROS) plays a pivotal role in cell death inducedby many stimuli. Lowlevels of ROS occur normally withincells due to electrons escaping from the electron trans-port chain and reacting with oxygen molecules. However,

when cells are exposed to certain toxic stimuli, such asTNF- or hydrogen peroxide, the levels of intracellularROS rapidly increase due to mitochondrial dysfunction.These ROS cause oxidative damage to the cell, which canpotentially result in death of the cell either by apopto-sis or necrosis depending on the levels of ROS generated.Several studies have shown that sHsps, especially Hsp27and -B-crystallin, can protect against ROS generatedthrough TNF- stimulation as well as oxidative stressinduced by hydrogen peroxide and menadione.92 Thesediscoveries led to the hypothesis that these sHsps couldfunction as inhibitors of ROS action, by modulating andmaintaining the redox parameters within cells. Although

Hsp27 is devoid of any endogenous ROS detoxifying ac-tivity, it can increase intracellular levels of glutathione,93

a tripeptide with numerous functions within cells, in-cluding ROS detoxification and regulation of cell death.Interestingly, treatment of cells with agents that lowerlevels of glutathione, enhance the induction of stress pro-teins including Hsp27,94 suggesting that the expressionof sHsps acts as a bufferingsystem to prevent theoxidationof proteins such as is normally seen when intracellular lev-els of ROS increase. This ability of sHsps to increase andmaintain glutathione in a reduced form correlates with anincrease in glucose-6-phosphate dehydrogenase activity,

an enzyme involved in the ROS-glutathione pathway.95

Hsp27 in tumorigenesis

Dysregulation of apoptosis is well known to play a rolein many diseases, including degenerative disorders andmany cancers. In this regard, the intracellular levels of

molecules such as Hsp27 that are involved in the regula-tion of apoptosis are crucial for maintaining the balancebetween cell death and cell survival within an organism.

Although Hsp27 displays constitutive expression in re-stricted tissues it is apparent that variations in its ex-pression levels could have potentially deleterious effects.Higher levels of Hsp27 expression, relative to those innon-transformed cells, are commonly detected in a vari-ety of different cancers including breast,96,97 prostate,98

gastric99 and ovarian100 cancers as well as Hodgkinsdisease.101 Since dysfunctional apoptotic signalling iscommonly found in cancer cells where it plays an im-

portant role in tumour initiation and progression, in-creased levels of Hsp27 expression may render tumoursmore resistant to host defence mechanisms as well as manycommonly used chemotherapeutic agents. Indeed, sev-eral studies point to the ability of Hsp27 to increase themetastatic potential of tumors cells in nude mice as wellas enhancing their resistance to therapy.102,103

In spite of these observations, attempts to correlatethe levels of Hsp27 in tumor cells with clinical prog-nosis and progression of the tumor have proved difficultowing to contradictory results. For example, the expres-sion of Hsp27 in breast cancer is correlated with thatof the estrogen receptor and is associated with small tu-mour size and low proliferative index, but its expressionin node negative cases is linked with short disease-freesurvival.101,104 Even so, although notuniversal, in thevastmajority of cases the expression of Hsp27 within tumoursis correlated with poor prognosis and short-disease freesurvival.

Furthermore, what may be more worthy of clinical noteis the fact that several of the commonly used chemother-apeutic drugs such as doxorubicin can modulate and in-crease expression of Hsp27 and other Hsps. Therefore, ifthese drugs are not administered at levels high enoughto achieve apoptosis of the tumor cells they may actu-

ally enhance the ability of the cells to resist conventionalchemotherapeutic drugsthroughthe upregulation of anti-apoptotic factors like Hsp27.105

Conclusion

Without doubt the expression of Hsp27 serves as a protec-tive mechanism to increase cellular survival during timesof stress. The precise mechanism of how Hsp27 mediatesthis protection is highly complex, as demonstrated by thevariety of different roles proposed for the protein. It is

apparent from these observations that the functioning of



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Hsp27 is a highly adapted and dynamic one with post-translational modifications playing an important role. Ex-pression of Hsp27 is only transiently induced in responseto the stress event, after which expression levels fall dras-tically, thus allowing only for overexpression when itscytoprotective properties are required. Based on the evi-dence presented in this review it is plausible to suggestHsp27 has two distinct roles during stress: (1) to maintainthe normal function of cells through interaction with andstabilization of the cytoskeleton and by facilitating therepair or removal of damaged proteins and (2) to preventapoptosis by interfering with caspase activation throughan ability to sequester cytochrome cand pro-caspase-3 andalso by acting as a redox modulator.

Undoubtedly, full elucidation of the role of Hsp27 willhave many potential uses as therapies for several ailments.Already gene therapy experiments have proven the use-

fulness of using exogenous Hsp27 for therapeutic benefit.Wagstaff and co-workers have demonstrated that the de-livery of exogenous Hsp27 to cultured neuronal cells byuse of HSV-based vectors,106 provides protection from theinductionof apoptosisby various stimuli. Since many neu-rodegenerative diseases such as Alzheimers Disease andParkinsons Disease result from excessive death of neu-ronal cells, some of which is apoptotic in nature, the useof gene therapy to deliver exogenous Hsp27 may help toimprove the prognosis for these diseases. Similar effectshave been demonstrated for cardiac cells challenged withhypoxia and thermal stress.107 Alternatively several dif-ferent approaches are being developed to upregulate theendogenous levels of Hsp27 and other Hsps. These in-clude the induction of Hsps by the use of cytokines suchas IL-2 and the use of pharmacological agents that couldbe given to patients to induce enhanced Hsp expressionin a non-stressful manner.108

Overexpression of Hsp27 has been demonstrated in avariety of cancerous cells where it has been seen to en-hance the tumour-forming ability of these cells. In manycases the expression of Hsp27 can inhibit the effectivenessof cytotoxic drugs in eliminating these tumours. There-fore, the use of technologies to eliminate the expressionof Hsp27 may have potential uses for the treatment of

Hsp27-overexpressing cancers. Indeed, experiments haveshown that the use of Hsp70 antisense technology inHsp70-overexpressing tumours causes these cells to spon-taneously undergo apoptosis.109 It is possible that the useof Hsp27 antisense in combination with cytotoxic drugsmay also prove a useful approach in the treatment of can-cers to induce apoptosis. Alternatively, as we begin tofully comprehend how Hsp27 mediates its anti-apoptoticability it may become possible to systematically designpharmaceutical compounds that bind to the regions ofHsp27 responsible for its interactions with cytochrome cor procaspase-3, thereby alleviating Hsp27s ability to

inhibit apoptosis.

Acknowledgments

The authors research is supported by generous fundingfrom Enterprise Ireland, European Commission, HealthResearch Board of Ireland, Higher Education Author-

ity, Irish Heart Foundation and the Millennium ResearchFund of NUI, Galway.

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