Selective protection by hsp 70 against cytotoxic drug-, but not Fas-induced T-cell apoptosis

doi:10.1046/j.1365-2567.1999.00758.x

Journal List > Immunology > v.97(1); May 1999

Immunology. 1999 May; 97(1): 36–44.

doi: 10.1046/j.1365-2567.1999.00758.x.

PMCID: PMC2326807

Selective protection by hsp 70 against cytotoxic drug-, but not Fas-induced T-cell apoptosis

E M CREAGH and T G COTTER

Tumour Biology Laboratory, Department of Biochemistry, University College Cork, Cork, Ireland

Correspondence: Professor T. G. Cotter, Department of Biochemistry, Lee Maltings, Prospect Row, Cork, Ireland.

Received October 20, 1998; Accepted January 7, 1999.

This article has been cited by other articles in PMC.

Abstract

The phenomenon of heat-shock (HS) protection to many cytotoxic insults has previously been described; however, the specific molecular mechanism underlying this HS-mediated protection remains undefined. To gain insight into this protective mechanism, heat-shocked Jurkat T cells were treated with a range of cytotoxic agents. Those against which HS conferred protection (camptothecin and actinomycin D) were compared with agents against which HS showed no protective effect (anti-Fas monoclonal antibody (mAb)). Reactive oxygen species (ROS) production was found to be an event common to apoptosis induced by camptothecin and actinomycin D, whereas Fas-mediated apoptosis was shown to occur via a ROS-independent mechanism. The selective protection observed against these agents was found to be mimicked by pretreatment with antioxidant compounds. Furthermore, this antioxidant protection appears to be occurring downstream of ROS production. Experiments were extended using heat-shock protein (hsp) 70 gene-transfected Jurkat T cells to confirm that the protective effects observed were caused by hsp 70 synthesis rather than any other cellular response to HS. Bcl-2 expression levels were also examined to determine whether any correlation existed between Bcl-2- and hsp 70-mediated protection.

INTRODUCTION

Apoptosis is a highly regulated process which involves the activation of an endogenous suicide programme.¹ First described by Kerr et al. in 1972,² apoptosis is characterized morphologically by chromatin condensation, cell shrinkage, membrane blebbing and the formation of apoptotic bodies. The CD95 (Fas/Apo-1) receptor/ligand system is one of the best-defined apoptotic pathways to date. This system plays a major role in the deletion of cells in the immune system, particularly during the maturation of T cells and cytotoxic T lymphocyte (CTL)-mediated cytotoxicity of target T cells.³ Cytotoxicity by a range of chemotherapeutic drugs including camptothecin (Camp)² and actinomycin D (Act D) is also mediated by the induction of apoptosis.⁴ The precise mechanism by which these chemotherapeutic drugs induce apoptosis remains to be fully elucidated. A role for the Fas system in drug-induced apoptosis has recently been suggested;⁵ however, work from our laboratory demonstrates that recruitment of the Fas receptor/ligand system is not a necessary requirement for drug-induced apoptosis.⁶

Damage to biological systems caused by the generation of reactive oxygen species (ROS) is often referred to as ‘oxidative stress’. The term ROS is a collective one that includes free radicals such as the superoxide anion (O⁻₂), hydroxyl radical (OH·) and also some non-radical derivatives of oxygen, such as hydrogen peroxide. Antioxidant compounds have been widely reported to protect against, or delay apoptosis induced by a wide range of stimuli.⁷ Recent evidence has proposed oxidative stress as a potential common mediator of apoptosis.⁸ Camptothecin and actinomycin D have been shown to rapidly produce ROS during the induction of leukaemic cell apoptosis.⁹^,¹⁰ However, several agents, including Fas, have been reported to induce apoptosis independently of ROS production.¹¹^,¹² A recent report demonstrates that Fas-mediated induction of apoptosis in Jurkat T lymphocytes results in a rapid loss of reduced glutathione (GSH), but no accompanied increase in ROS levels is detectable.¹³

The heat shock proteins (hsps) represent a family of proteins containing both constitutively expressed and stress inducible members. A mild heat shock (HS) treatment to cells results in a transient state of cellular thermotolerance which correlates quantitatively with the extent of hsp synthesis.¹⁴ Heat shock protein 70 (hsp 70), one of the most abundant hsps, has the highest correlation between thermotolerance and synthesis during and following HS treatment.¹⁵ Heat shock has been reported to increase resistance to apoptosis induced by ethanol, hydrogen peroxide (H₂O₂), tumour necrosis factor-α (TNF-α) and a number of chemotherapeutic agents.¹⁶^–¹⁸ Many, if not all, of these treatments have been found to cause oxidative stress within cells.¹⁹ Interestingly, a recent report has suggested that hsp synthesis, while inducing thermotolerance, can actually enhance Fas-mediated apoptosis.²⁰ Little is known about the requirement for, or mechanism of, hsp inhibition of apoptosis. The Bcl-2 protein, a major regulatory protein in the control of apoptosis,²¹ has been proposed to inhibit apoptosis via an antioxidant pathway.²²^,²³ Bcl-2 has previously been shown to inhibit heat-induced death²⁴ and increased Bcl-2 expression has recently been demonstrated following HS in a number of cell lines.²⁵

This present study was conducted to determine whether heat-inducible hsps selectively protect against agents that induce oxidative stress during the induction of apoptosis. The cytotoxic agents chosen for this study were Camp, Act D and Fas, based on their reported ROS-dependent and -independent mechanisms of action.⁹^,¹⁰^,¹² The putative role of Bcl-2 in this HS-mediated inhibition of apoptosis was also investigated. Our results indicate that heat-induced hsps selectively inhibit apoptosis induced by ROS-producing agents. Protection against ROS-producing agents is demonstrated to occur at maximum levels of hsp synthesis. In addition, hsp synthesis directly protects against the exogenous addition of hydrogen peroxide as an apoptotic signal to these cells. In stark contrast, no reduction in Fas-mediated apoptosis is observed. Hsp 70 gene-transfected Jurkat cells were used to indicate that the selective inhibition of apoptosis being observed was caused by hsp synthesis rather than any other cellular response to HS. The protective mechanism is demonstrated to be independent of Bcl-2 upregulation.

MATERIALS AND METHODS

Cell culture and transfection

The human leukaemic T-cell lymphoblast line, Jurkat, was maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 mm glutamine and 1% penicillin–streptomycin. Jurkat cells were transfected with the phβApr-1-neo (vector-transfected) and phβApr-hsp 70 (gene-transfected) plasmids by electroporation. The plasmids phβApr-1-neo²⁶ and phβApr-hsp 70²⁷ were a gift from L. Kedes and Ch. Angelidis, respectively. Electroporation was carried out at a voltage of 300 V and capacitance of 960 μF using a BioRad (Hemel Hempstead, UK) Gene-Pulser and capacitance extender. Transfectants were selected and maintained in RPMI supplemented with geneticin (G418). Cells were maintained at 37° in a humidified 5% CO₂ atmosphere. All reagents, unless otherwise stated, were obtained from Gibco (Paisley, UK).

Heat-shock treatment

Cells were suspended at 5 × 10⁵ cells/ml in closed Eppendorf tubes (1·5 ml). The tubes were suspended in a water bath for 1 hr at 42°, centrifuged at 200 g for 5 min in a sorvall MC12 V minifuge (DuPont Ltd, Hertfordshire, UK) and resuspended in fresh medium before returning to a 37° incubator for a minimum of 8 hr to allow for maximum hsp synthesis.

Induction of apoptosis

Jurkat cells (5 × 10^5/ml) were seeded in six-well plates (Nunc, Paisley, UK) and exposed to various cytotoxic agents: Camptothecin (15 μg/ml) (Sigma, Poole, UK), Actinomycin D (15 μg/ml) (Sigma) and the anti-Fas immunoglobulin M (IgM) antibody (300 ng/ml) (Upstate Biotech., Lake Placid, NY), and incubated for 6 hr. Various concentrations of hydrogen peroxide (50–330 μm) (BDH, Poole, UK) were incubated for 16 hr at 37°.

Assessment of apoptosis

Cell number was assessed using a Neubauer haemocytometer and viability was determined by the ability of cells to exclude trypan blue (Sigma).Two methods were used to assess viability: morphology and Terminal dUTP Nick End-Labelling (TUNEL) assay.

Morphology Cells (80 μl, 5 × 10⁵/ml), in their suspension media, were cytospun onto slides using a Shandon (Cheshire, UK) cytospin 2 at 500 rpm for 2 min. Slides were air-dried, fixed and stained using the Rapi-Diff II staining kit (LanganBach Services, Wicklow, Ireland). Apoptotic cells were identified as described previously.²⁸

TUNEL assay: in situ nick end labelling assay The DNA in individual cells was labelled with exogenous terminal deoxynucleotidyl transferase (TdT) using a modification of a previously described method.²⁹ Briefly, cells (5 × 10⁵/ml) were fixed in 1% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS) for 15 min at 4°, washed in PBS and stored in a 70% ethanol solution at −20° overnight. After rehydration in PBS, the cell pellets were resuspended in 50 μl of the elongation solution (0·1 mol/l sodium cacolydate (pH 7·0), 0·1 mol/l dithiothreitol (DTT), 0·05 mg/ml bovine serum albumin (BSA), 5 U of TdT and 0·5 nmol/l biotin dUTP) and incubated for 1 hr at 37°. After washing in PBS, the cells were resuspended in 100 μl of staining buffer (saline–sodium citrate buffer containing 0·1% Triton-X-100, 5% w/v non-fat dry milk and avidin–fluoroscein isothiocyanate (FITC) 2·5 μg/ml) and incubated at room temperature in darkness for a further 30 min. Cell fluorescence was measured using CellQuest software on a fluorescence-activated flow cytometer (FACscan; Becton Dickinson, CA.) with excitation and emission settings of 488 and 600 nm, respectively. Bio-16-dUTP and the TdT enzyme were obtained from Boeringer (Mannheim, Germany). All other chemicals were obtained from Sigma (Poole, UK).

Western blotting

Cells were centrifuged at 200 g for 5 min and lysed in 20 μl suspension buffer (0·1 m NaCl, 10 mm HEPES (pH 8·0), 500 mm sucrose, 1 mm ethylenediamine tetra-acetic acid (EDTA) (pH 8·0), 1 μg/ml aprotinin, 0·15 mm spermine, 0·2% Triton-X-100 and 0·2 mm phenylmethylsulphonyl fluoride (PMSF)). An equal volume of 2× sodium dodecyl sulphate (SDS) gel loading buffer (100 mm Tris–HCl (pH 6·8), 200 mm DTT, 4% SDS, 0·2% bromophenol blue and 20% glycerol) was added to samples, boiled for 10 min and loaded on 10% SDS-polyacrylamide gel, electrophoresed and blotted onto BioRad nitrocellulose membrane. After neutralisation with 5% Blotto (5% non-fat dried milk) in PBS, the membranes were incubated for 2 hr with the relevant primary antibody. The rat antihuman HSP 90, murine anti-HSP 70 and murine anti-HSP 27 monoclonal antibodies (mAb) were incubated at a 1/1000 dilution (Stressgen, York, UK) and murine anti-β-Actin (Sigma) at a 1/5000 dilution. The Bcl-2 oncogene product was detected using the murine anti-Bcl-2 mAb (Dako, Glostrup, Denmark) at a 1/50 dilution. After washing in 5% non-fat dried milk, secondary antibodies (peroxidase-conjugated antimouse immunoglobulins (Dako) and antirat IgG (Sigma)) were incubated for a further hour, both at a 1/500 dilution. Bands were detected using the enhanced chemiluminescence (ECL) system (Amersham, Amersham, UK).

Measurement of intracellular peroxide levels

Peroxide levels were assessed using a method previously described.⁷ Briefly, cells (5 × 10^5/ml) were incubated with 5 μm dichlorofluorescein diacetate (DCFH/DA) (Molecular Probes, Leiden, The Netherlands), made as a 10-mm stock in dimethyl sulphoxide (DMSO), for 1 hr at 37°. Cells were treated with apoptosis-inducing agents either before or during this 1 hr incubation period (depending on the time point at which the peroxide measurement was to be made). Peroxide levels were measured using a FACScan flow cytometer with excitation and emission settings of 488 and 530 nm, respectively.

Measurement of intracellular superoxide anion levels

Superoxide anion levels were measured using an adaptation of the method employed by Rothe and Valet.³⁰ The dye hydroethidine (Molecular Probes) was used, which is oxidised by the superoxide anion within the cell to produce ethidium bromide which fluoresces when it intercalates into DNA. Cells (5 × 10⁵/ml) were incubated with 10 μm hydroethidine (made as a 10-mm stock in DMSO) for 15 min at 37°. Cells were treated with apoptosis-inducing agents either before, after or during this 15 min incubation period. Intracellular superoxide anion levels were assessed by measuring the fluorescence due to ethidium bromide using the FACScan flow cytometer with excitation and emission settings of 488 and 600 nm, respectively.

Treatment with antioxidants

Antioxidants used were: 2·5 μm pyrrolidinedithiocarbamate (PDTC) (Sigma) and 0·3 mm phenanthroline (Phen) (Reidel de Haen, Seelze, Germany). PDTC was prepared prior to use in distilled water as a 1-m stock, Phen was prepared in ethanol as a 1-m stock and was stored at −20°. Cells were incubated with the antioxidants 10 min prior to addition of the apoptosis-inducing agent.

RESULTS

Hsp 70 and 27 synthesis following heat-shock treatment

The synthesis of hsps following heat-shock treatment has already been documented in numerous cell lines.³¹ The initial experiment in the present study focused on determining the optimum conditions for hsp induction. Cells were heat shocked (1 hr at 42°) and allowed to recover at 37° for varying time points (0–24 hr). Hsp expression levels were observed by western blot (Fig. 1). Hsp 27 expression is evident after 8 hr and hsp 70 expression is maximal at this time interval. All further experiments involving heat-shock-treated cells were therefore left to recover for 8 hr prior to treatment.

Figure 1

Western blot showing increase in hsp expression following heat shock (HS). Cells were HS for 1 hr at 42°, and then allowed to recover at 37° for 2–24 hr as indicated. Lane 0 indicates non-heat-shocked (NHS) cells. Hsp 70 expression (more ...)

Heat-shock-treated cells show selective resistance to camptothecin and actinomycin D treatments

To observe the protective role with which the hsps have been associated, the cytotoxic effects of three apoptosis-inducing agents (Camp, Act D and anti-Fas mAb) were examined using HS-treated and untreated cells. HS-treated cells exhibited a marked resistance to Camp- and Act D-induced apoptosis, whereas no reduction in apoptosis was observed in cells treated with anti-Fas mAb (Fig. 2a, b).

Figure 2

The effect of HS on the cytotoxicity of chemotherapeutic agents. Cells (HS and NHS) were given an 8-hr recovery period prior to a 6-hr exposure to Camp (15 μg/ml), Act D (15 μg/ml) and Fas (300 ng/ml). (a) Percentage apoptosis was estimated (more ...)

Significant ROS production following camptothecin/actinomycin D addition

To investigate the hypothesis that the selective protection to apoptosis demonstrated may be related to the oxidative status of the cells, the ROS (superoxide and peroxide) levels in cells treated with Camp, Act D and anti-Fas mAb were studied. Significant peroxide production was observed following Camp and Act D treatment (Fig. 3a, b), while ROS levels following Fas treatment remained basal (Fig. 3c), suggesting that Camp and Act D may be inducing apoptosis via a ROS-dependent pathway, and that the hsps could be protecting cells by interfering with this pathway.

Figure 3

Superoxide and peroxide levels over a 2-hr time course following the addition of (a) Camp (15 μg/ml), (b) Act D (15 μg/ml) and (c) Fas (300 ng/ml). Points plotted were mean values of FL1 fluorescence (peroxide) and FL2 flourescence (superoxide) (more ...)

Heat-shock treatment reduces hydrogen peroxide-induced cytotoxicity

Untreated and HS-treated cells were incubated with varying concentrations (50–330 μm) of hydrogen peroxide to determine whether hsps are protective against increased levels of intracellular peroxide levels. HS-treated cells show a marked reduction in apoptosis (Fig. 4), suggesting that the hsps may be protecting against Camp and Act D cytotoxicity by a mechanism which specifically interferes with their induction of peroxides.

Figure 4

Heat-shock protection to increasing concentrations of H₂O₂ following 16 hr incubation with the oxidant. Percentage apoptosis was estimated using morphological criteria. Results are means ± SE values from three independent experiments.

Antioxidants also reduce the cytotoxicity of Camp and Act D

To investigate the involvement of the increased ROS levels induced by Camp and Act D to their overall toxicity, cells were pretreated with the antioxidants phenanthroline (Phen) and pyrrolidinedithiocarbamate (PDTC) prior to treatment with the three cytotoxic agents: Camp, Act D and Fas. These antioxidants reduced the cytotoxicity of both Camp and Act D to a similar extent as that of heat-shock treatment and as expected, conferred no reduction to Fas-induced apoptosis. Results are shown by morphological analysis (Fig. 5a) and the TUNEL assay (Fig. 5b).

Figure 5

Antioxidant protection to ROS-producing cytotoxic agents. Cells were incubated with Phen (0·3 mm) and PDTC (2·5μm) 10 min prior to the addition of Camp (15 μg/ml), Act D (15 μg/ml) and Fas (300 ng/ml). Antioxidant (more ...)

Antioxidant protection is not a result of decreased ROS production

The ROS levels of cells treated with antioxidants followed by the addition of Camp and Act D were measured, revealing that ROS remained at the same elevated levels (Fig. 6), even though these cells are protected against apoptosis. The ROS levels of heat-shocked cells after addition of these cytotoxics were also unchanged (data not shown). This observation implies that both the antioxidants and hsps are exerting their protective effects downstream of ROS production and blocking their deleterious effects.

Figure 6

Antioxidant protection to Camp and Act D does not involve reduction in ROS levels. Peak peroxide levels following 20 min Camp and 5 min Act D incubations were compared to those pretreated with Phen (0·3 mm). (I) 1 Control, 2 Camp, 3 Phen- and (more ...)

Hsp 70 gene-transfected cells are protected against ROS-mediated apoptosis

To confirm that the protective effects shown by heat-shock-treated cells in this study are caused by the synthesis of hsps and not to some other HS response, Jurkat cells stably transfected with the hsp 70 gene were subjected to similar experimental conditions. These tranfectants express hsp 70 without HS treatment. Increased levels of hsp 70 (the inducible form of the protein, also referred to as hsp 72) in the gene-transfected cells are shown by western blot (Fig. 7a). Morphological analysis of control, vector- and gene-transfected cells treated with the ROS-inducing agents Camp, Act D, and hydrogen peroxide revealed that hsp 70 gene-transfected cells showed increased resistance to apoptosis (Fig. 7b) to the same extent as HS-treated cells, suggesting that hsp 70 is likely to be the major protective influence in the HS cells.

Figure 7

(a) Evidence for hsp 70 gene overexpression in transfected cells. Western blots of lysates from control Jurkat cells (N), HS Jurkat cells (H), vector (V)- and gene (G)-transfectants. Equal lane loading was verified by probing for β-actin. (b) (more ...)

Hsp 70-mediated protection occurs independently of Bcl-2 upregulation

To determine whether there was any relationship or correlation between Bcl-2 expression, hsp 70 and/or the thermotolerant state in the inhibition of apoptosis, Bcl-2 levels in cells from untreated, HS-treated, vector- and gene-transfected cells were observed by Western blot (Fig. 7c). Results show that neither hsp 70 overexpression nor HS treatment cause an increase in Bcl-2 expression, suggesting that hsp 70 may be protecting by a Bcl-2-independent mechanism. The expression levels of Bcl-XL, Bax and Bak were also examined for up/downregulation by Western blot; as for Bcl-2, no change in expression was observed (data not shown).

These results strongly suggest that hsp 70 can protect cells from cytotoxic agents which induce apoptosis by biochemical pathways involving ROS, and that the mechanism of protection is independent of Bcl-2 upregulation.

DISCUSSION

The ability of hsps to inhibit apoptosis induced by a diverse range of stimuli has previously been reported in a number of cell systems.¹⁷^,¹⁸ The specific molecular mechanisms underlying this heat shock mediated protection, however, remain undefined. HS treatment has been shown to inhibit apoptosis induced by the addition of exogenous ROS such as H₂O₂ in many cell types.³²^,³³ A recent report has demonstrated that hsp 70 overexpression offers thermoprotection, but enhances Fas- and T-cell receptor (TCR)-mediated cell death,²⁰ both of which are ROS independent.¹²^,³⁴ Conversely, TNF-α receptor-mediated apoptosis can be inhibited by hsp 70.¹⁸ TNF-α, however, relies on a ROS burst to activate phospholipase A₂, thereby triggering its apoptotic cascade.³⁵ Many diversly acting cytotoxic agents, including Camp and Act D, produce ROS during their course of action.⁹^,¹⁰ The Fas receptor, a member of the TNF receptor superfamily, is structurally homologous to the TNF-α receptor.³⁶ Interestingly, overexpression of Bcl-2 has been reported to impair both TNF-α- and chemotherapeutic-mediated cytotoxicity;³⁷^,³⁸ however, there is much evidence implicating a Bcl-2-independent pathway for Fas-mediated cell death.³⁹

Our findings demonstrate that HS-induced hsp expression selectively inhibits apoptosis induced by the ROS-producing agents Camp, Act D and the oxidant H₂O₂. The heat-inducible hsps, however, do not inhibit Fas-mediated apoptosis. Cells pretreated with the antioxidants phenanthroline and PDTC show increased resistance to Camp- and Act D-induced apoptosis, suggesting a major role for ROS in their cytotoxicity. In addition, the level of resistance to apoptosis given to cells by these antioxidants is analogous to that of HS-treated cells. Antioxidants may function by either directly scavenging ROS or, alternatively, by blocking the effect of ROS production, thus relieving cellular oxidative stress.⁴⁰ Both phenanthroline and PDTC are reported to have metal-ion-chelating properties.⁴¹ These chelators function by removing free transition metal ions, thereby minimizing the amplification of ROS toxicity within the cell.⁴² Our results demonstrate no decrease in ROS levels following pretreatment with antioxidants or HS, suggesting that the protective effects of the hsps and antioxidants are occurring downstream of ROS production. Phenanthroline has previously been proposed to have an antioxidant function that inhibits caspase 1 in vitro by contaminating metal-catalysed oxidation of an essential thiol.⁴³

In addition to hsp synthesis, HS inhibits the synthesis of most other polypeptides, affects cellular functions and may cause organelle modification.¹⁴ Jurkat cells transfected with the hsp 70 gene allowed confirmation that the protective phenomenon occurring following HS treatment was specifically caused by hsp synthesis, and not any other heat-induced cellular perturbation. Resistance to apoptosis (induced by Camp, Act D, Fas and H₂O₂) in hsp 70 transfectants was comparable to those obtained using HS cells, suggesting that the protection observed following HS treatment is largely caused by the hsp 70 protein. Bcl-2 involvement in this hsp 70-mediated mechanism of protection was investigated, as there is direct evidence for the antioxidant function of Bcl-2 in protection against apoptosis²²^,²³ and correlations between HS and Bcl-2 have previously been reported.²⁴^,²⁵ Levels of Bcl-2 expression in both HS and hsp 70 transfectants were found to be identical to those in control Jurkat cells, indicating that the hsp 70-mediated inhibition of apoptosis, although appearing to occur via an antioxidant pathway, is independent of Bcl-2 upregulation.

This study suggests a novel function for the hsp 70 family in the inhibition of apoptosis-induced by oxidative stress. We have shown that the protective function of hsp 70 is specific to ROS-producing cytotoxic agents. The mechanism, by which hsp 70 increases resistance to oxidative stress-induced apoptosis, remains to be established. The elimination of Bcl-2 upregulation as a mediating factor in this protective mechanism suggested the cellular antioxidant defence system as a likely mediator in this hsp 70-mediated inhibition of apoptosis. The major endogenous antioxidant enzymes are catalase, superoxide dismutase and glutathione peroxidase, which serve to scavenge ROS generated during cellular metabolism.¹⁹ Hsp inducers, such as hyperthermic stress, have been demonstrated to simultaneously stimulate antioxidant enzyme activities and hsp expression in various cellular systems.⁴⁴ It is therefore possible that hsp 70 may (either directly or indirectly) enhance endogenous antioxidant enzyme levels/activities to produce the selective resistance to apoptosis observed by hsp 70 in this study. The observation that ROS levels remain elevated in resistant (HS and hsp 70 overexpressing) cells, however, makes the upregulation of this antioxidant defence system an unlikely mechanism for the protective effect being observed.

Our results suggest that the target for hsp 70-mediated inhibition of apoptosis is downstream of ROS production. Possibilities include the inhibition of caspase activity, a mechanism that has previously been proposed for the antioxidant phenanthroline.⁴³ Another study suggests that hsp 70 confers protection against increased intracellular ROS levels by increasing the cells’ tolerance threshold.⁴⁵ This suggestion is supported by the fact that release of the pro-inflammatory cytokines interleukin (IL)-1 and IL-6, which are secreted in response to oxidative stress,⁴⁶ is reduced in cells overexpressing hsp 70. Another possible target for the inhibition of apoptosis by hsp 70 may be the mitochondria. ROS have been reported to target these organelles,⁴⁷ causing disruption of the mitochondrial membrane potential (ΔΨ_m), leading to alterations in calcium homeostasis, protease activation, and apoptosis.³⁶

The constitutive hsp 70 family members are localised within different intracellular compartments, including the endoplasmic reticulum, mitochondria and nucleus. They are ATP-binding proteins that function as chaperones to facilitate protein assembly, folding and translocation.⁴⁸ Given the role of hsp 70 as a chaperone, it may be possible that the HS-induced hsp 70 is binding to agents/proteins that link ROS production to initiation of the apoptotic cascade, causing a block in the oxidative stress mediated pathway of apoptosis. The proteins with which hsp 70 may be associating are currently under investigation.

Acknowledgments

This work was supported by the Health Research Board of Ireland and Forbairt, Ireland. The authors are grateful to Professor Ch. Angelidis, University of Ioannia and Professor L. Kedes, University of Southern California for kindly sending the phβApr-hsp 70 and phβApr-1-neo plasmids, respectively. The authors are indebted to Drs S.L. McKenna, A. Samali and A. Gorman for their helpful advice during the course of this work. We are also grateful to Drs S.L. McKenna, A.J. McGowan and R. J. Carmody for the critical evaluation of this manuscript.

	Abbreviations
HS	heat shock
Camp	camptothecin
Act D	actinomycin D
ROS	reactive oxygen species
hsp	heat-shock protein
H₂O₂	hydrogen peroxide
TNF-α	tumour necrosis factor-α
Phen	phenanthroline
PDTC	pyrrolidinedithiocarbamate.

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