Tobacco smoke induces both apoptosis and necrosis in mammalian cells: differential effects of HSP70

Muriel Vayssier1, Nathalie Banzet1, Dominique François1, Kerstin Bellmann2, and Barbara S. Polla1,3

1 Laboratoire de Physiologie Respiratoire, Unité de Formation et de Recherche Cochin Port-Royal, 75014 Paris, France; 2 Diabetes Research Institute, Heinrich-Heine University, 40225 Düsseldorf, Germany; and 3 Environment and Health Program, Faculty of Medicine, University Hospital, 1211 Geneva 14, Switzerland

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Tobacco smoke (TS) has been implicated as a major risk factor in human pulmonary diseases including cancer. In this study, we used TS as a model of oxidative stress. TS-mediated oxidative stress has been shown to induce protein oxidation, DNA damage, and cell death. Here we investigated, in human and rodent cell lines, whether TS induces cell death by apoptosis or by necrosis. As described for classic oxidants, TS induced apoptosis at low concentrations and necrosis at higher concentrations. We have previously described the induction of heat shock (HS) protein (HSP) (in particular, HSP70) in human monocytes exposed to TS. HSP70 is implicated in the regulation of cell injury and cell death and, in particular, modulates apoptosis, as does the antiapoptotic oncoprotein Bcl-2. At both apoptotic and necrotic concentrations, TS induced a dose-dependent HSP70 expression, whereas Bcl-2 was induced only at necrotic concentrations. TS- or HS-induced HSP had no protective effects either on apoptosis or on necrosis, but HSP70 overexpression prevented TS-induced necrosis and consequently led to increased apoptosis. These results might reconcile the apparently contradictory data previously reported on the effects of HSP on apoptosis.

heat shock protein 70; Bcl-2

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

TOBACCO SMOKE (TS) has been implicated as a major risk factor in pulmonary diseases such as chronic bronchitis and emphysema, in carcinogenesis, and in cardiovascular disease (27). The toxicity of TS is due to a large variety of compounds, including nicotine, cadmium, benzo[a]pyrene, oxidants, and free radicals, that initiate, promote, or amplify oxidative damage (32). From this point of view, TS can be used as a model of exposure to environmental reactive oxygen species (ROS) or ROS inducers. TS-mediated oxidative stress has been shown to lead to protein oxidation, DNA damage, and cell death.

Pinot et al. (28) have previously described TS induction of stress proteins, including heat shock (HS) proteins (HSPs) and, in particular, the cytosolic, inducible 72-kDa HSP70 in human monocytes. Expression of HSP is a conserved, adaptive response to various stresses (e.g., thermal, oxidative, or infectious). HSPs act as molecular chaperones, contributing to the folding of nascent polypeptides and to protein transport and degradation. They protect cells and organisms from oxidative damage both in vivo and in vitro (17, 29, 31) and, more generally, prevent cell death (34, 37). In a number of in vitro and in vivo apoptotic models, HSP70 has been shown to act as a protective factor (10, 22, 26, 34, 37). However, these "protective" effects of HSP might also have deleterious effects by promoting carcinogenesis. Indeed, Seo et al. (35) have shown that transgenic mice overexpressing HSP70 on a lymphocyte-targeted promotor developed lymphoma. These data provide evidence for a causal relationship between prevention of apoptosis secondary to HSP70 overexpression and carcinogenesis. TS-induced overexpression of HSP70 could thus, by preventing apoptosis, promote carcinogenesis rather than exert any protective effect. A similar relationship between the prevention of apoptosis and subsequent carcinogenesis has also been established for the protooncoprotein Bcl-2, another major negative regulator of cell death, which can prevent cells from undergoing apoptosis induced by various stimuli (18, 21).

With respect to the role of HSPs in apoptosis, however, contradictory data are emerging. First, HSPs do not have the same protective power in all apoptosis models. Second, some studies (8, 24) show no protective effects of HSPs in apoptosis at all, even though these molecules are induced during apoptosis. Third, in some models, HSPs have been considered as apoptosis enhancers: overexpression of HSP70 enhances T-cell receptor/CD3- and Fas/Apo-1/CD95-mediated apoptosis in Jurkat T cells, and overexpression of HSP90 leads to increased tumor necrosis factor (TNF)-alpha - and cycloheximide-induced apoptosis of the human U-937 cell line (11, 23). These conflicting data on HSP functions in apoptosis have been tentatively explained by the differential mechanisms used by distinct cells to respond to different apoptosis-inducing stimuli.

Many of the chemical and physiological events capable of inducing apoptosis are also known to evoke oxidative stress. As a model for oxidative stress relevant to clinical pathology, we used in vitro exposure of TS to human and rodent (monocytic and pancreatic, respectively) cell lines. We examined whether TS induces cell death by apoptosis or necrosis and investigated the putative role of HSP70 in cell death mechanisms. If indeed HSP70 overexpression inhibited TS-induced apoptosis in vitro, it might be anticipated that HSP70 would contribute to TS-mediated carcinogenesis in vivo. Our data argue against this hypothesis and suggest that the contradictory data previously reported by others (23, 34) on the effects of HSPs on apoptosis could be explained by their differential effects on the two types of cell death, either apoptosis or necrosis.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cells

The human premonocytic line U-937 was maintained mycoplasma free in stationary suspension in RPMI 1640 medium (ICN Biomedicals; Costa Mesa, CA), containing 10% fetal calf serum (FCS), 25 mM HEPES, 1% glutamine, 25 µg/l of ampicillin, and 120 µg/l of penicillin (all from GIBCO, Egenstein, Germany). The rat insulinoma parental line RINm5F and the sublines R70/20 and RK/2, stably transfected with human hsp70 gene and with pZEM-neo eucaryotic expression vector, respectively, were cultured in RPMI 1640 medium supplemented with 10% FCS, 25 µg/ml streptomycin, 120 µg/ml penicillin, and 1% glutamine (all from GIBCO) (3). Cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2 in air.

HS

Exposure to HS was performed on U-937, RINm5F, R70/20, and RK/2 cells in the presence of 25 mM HEPES by incubating the cells in a water bath at 44°C for 30 min, as previously described (28). Cells were then allowed to recover at 37°C for 3 h before analysis. For HS pretreatment, U-937 cells were incubated at 44°C for 30 min and allowed to recover overnight before exposure to TS.

TS Exposure

TS exposure was performed as previously described (28). TS as intended here is both gas and particulate mainstream TS (i.e., smoke directly inhaled by smokers). Briefly, TS-bubbled phosphate-buffered saline (PBS) was generated by a peristaltic pump-smoke exposure machine (H. Borgwaldt RM1/G, Hamburg, Germany) (13, 28). This machine generates aqueous cigarette smoke fractions from standard research cigarettes (reference 2R1, University of Kentucky, Lexington) through a puffing mechanism at 1 puff/min (1 puff = ~35 ml of mainstream smoke). In the experiments presented here, the smoke of 1 cigarette corresponds to 10 puffs and is bubbled into 5 ml of PBS. Cells (0.25 × 106/ml) were exposed to TS concentrations ranging from 0.03 to 0.96 puff/ml for the indicated time (4 or 16 h for protein expression analysis, 6 or 16 h for apoptosis and necrosis analyses).

Flow Cytometry Analysis of Cell Death by Apoptosis and Necrosis

Necrosis and apoptosis are two distinct mechanisms of cell death that differ from each other by morphological, biochemical, and cytological characteristics. During apoptosis, cells display rearrangements in membrane phospholipids. In particular, phosphatidylserine (PS), which in normal cells is located in the inner cytoplasmic membrane, becomes exposed on cell surfaces, whereas the plasma membrane remains impermeable to dyes such as propidium iodide. In contrast, during necrosis, cells become swollen and the membrane is rapidly disrupted. In our experiments, apoptosis was detected with annexin V, which has high affinity for negatively charged phospholipids such as PS, conjugated to FITC. The simultaneous use of the DNA stain propidium iodide, which is normally excluded from intact and apoptotic cells, is able to detect the necrotic cells from within the annexin V-positive cell cluster. Cells exclusively positive for annexin V were considered as undergoing apoptosis, whereas cells positive for both propidium iodide and annexin V were considered as necrotic (9). Six or sixteen hours after TS exposure, U-937, RINm5F, R70/20, and RK/2 cells were washed, stained with annexin V and propidium iodide in HEPES buffer as described by the manufacturer (Blender MedSystems, Vienna, Austria), and analyzed by flow cytometry with an EPICS Elite flow cytometer (Coulter, Miami, FL) equipped with a single 488-nm argon laser. In all cases, a total of 5,000 cells/sample were analyzed in list mode for green fluorescence through a 525-nm filter and for red fluorescence through a 575-nm filter. All data were analyzed with Elite software version 4.02.

Electron Microscopy

After TS treatment, U-937 and RINm5F cells were fixed in suspension (106 cells) with 2.5% glutaraldehyde in 0.1 M PBS, pH 7.4, for 10 min at 4°C and as a pellet for 2 h. After being washed in PBS, ultrathin sections were cut and counterstained as previously described (31), then examined in a Philips EM-300 electron microscope operating at 60 kV.

HSP70 and Bcl-2 Expression

U-937, RINm5F, R70/20, and RK/2 cells were exposed to TS concentrations ranging from 0.06 to 0.96 puff/ml for 4 or 16 h or to HS for 30 min and allowed to recover for 3 h as a positive control of HSP70 expression. Aliquots corresponding to 1 × 106 cells were washed with PBS before being fixed for 10 min in 3% paraformaldehyde. After the cells were washed, they were incubated with 50 µl of 0.6% saponin (Sigma, St. Louis, MO) in PBS containing 1% BSA (PBS-BSA; Sigma) to permeabilize cell membranes. Intracellular HSP70 was detected with the anti-human antibody stained against the cytosolic, inducible HSP70 (Stressgen, Victoria, BC, Canada) at a dilution of 1:100 in PBS-BSA. A negative isotype-matched control IgG1 antibody (Dako, Glostrop, Denmark) was used to determine nonspecific binding. Unbound antibodies were removed by washing twice in PBS followed by staining with rabbit anti-mouse FITC (1:30 dilution, Dako) for 10 min in the dark. In U-937 cells, Bcl-2 was analyzed with the anti-human Bcl-2-FITC antibody (Dako) with a direct immunofluorescence technique at a dilution of 1:10 and compared with an appropriate isotype-matched FITC-conjugated control. Analysis was performed by flow cytometry (EPICS Elite).

Statistical Analysis

Data are presented as means ± SE. Statistical analysis was performed with the Mann-Whitney U-test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

TS Induced Apoptosis at Low Concentrations and Necrosis at Higher Concentrations in U-937 Cells

Apoptosis and necrosis were both detected after 16 h of exposure to TS. The apoptotic stage was evidenced by binding of annexin V to externalized PS and necrosis by propidium iodide. In our experiments, we considered live cells as annexin V(-), propidium iodide(-); apoptotic cells as annexin V(+), propidium iodide(-); and necrotic cells as annexin V(+/-), propidium iodide(+). As shown in Fig. 1, U-937 cells exposed to TS underwent cell death by either apoptosis or necrosis depending on the concentration of TS applied. Apoptosis increased from 0.03 puff/ml, with a maximum of apoptotic cells at 0.12 puff/ml. At concentrations >0.12 puff/ml, cell death by apoptosis decreased, whereas cell death by necrosis increased to reach 100% necrotic cells for concentrations >0.48 puff/ml. To confirm these results with another approach to assess cell death, we performed electron microscopy on U-937 cells exposed to low and high concentrations of TS (Fig. 2). U-937 cells exposed to low concentration of TS (0.12 puff/ml) showed morphological changes typical for apoptosis, including disintegration of the nucleolus along with preservation of plasma membrane, as well as mitochondrial swelling (Fig. 2B). When TS concentration increased (0.48 puff/ml), we observed intense vacuolization of U-937 cells, loss of plasma membrane integrity, and condensation of nuclear chromatin with a typical crescentlike appearance (Fig. 2C).


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Fig. 1.   Tobacco smoke (TS) induces apoptosis at low concentrations and necrosis at higher concentrations in U-937 cells. U-937 cells were exposed to TS doses ranging from 0.03 to 0.96 puff/ml for 16 h. Apoptotic cells were then detected by the binding of externalized phosphatidylserine with annexin V, whereas necrotic cells were detected by uptake of fluorescent cationic dye propidium iodide by using a cytofluorimetric approach. Values are means ± SE of %apoptotic or %necrotic cells; n = 4 experiments. Statistical analysis was performed by comparing TS-treated cells at each concentration with nontreated control cells for both apoptosis and necrosis.


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Fig. 2.   Morphological analysis of effect of TS on U-937 cells. A: nontreated U-937 cells display euchromatin and heterochromatin (×9,000). B: in U-937 cells exposed to 0.12 puff/ml of TS for 16 h, chromatin condensed around periphery of nucleus, nucleolus disintegrated, and mitochondria became swollen, whereas plasma membrane was preserved (×14,000). C: after treatment with 0.48 puff/ml of TS for 16 h, there was loss of plasma membrane integrity and intense vacuolization along with chromatin condensation (×9,000).

TS Induced a Concentration-Dependent Expression of Antiapoptotic Proteins HSP70 and Bcl-2 in U-937 Cells

HSP70 expression in U-937 cells exposed to TS. With the use of biometabolic labeling and Western blotting, Pinot et al. (28) previously reported that TS induces HSP in human monocytes. Because HSP70 prevents apoptosis under a number of conditions, it was of interest to examine whether TS, which induced apoptosis in U-937 cells, also induced, in parallel, antiapoptotic proteins such as HSP. We thus investigated HSP70 expression in U-937 cells in response to TS. U-937 cells were exposed to TS concentrations ranging from 0.06 to 0.96 puff/ml for 4 or 16 h. Intracellular HSP70 levels were analyzed in permeabilized cells by flow cytometry (Fig. 3A). Background levels of intracellular HSP70 were detected in nonstressed cells. Four hours of TS exposure induced the synthesis of the inducible HSP70 as measured by a dose-dependent increase in the percentage of cells expressing HSP70 (Fig. 3A). Similar levels of induced HSP70 were still detectable 16 h after exposure to TS (data not shown).


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Fig. 3.   TS induces dose-dependent expression of heat shock (HS) protein 70 (HSP70; A) and Bcl-2 (B) in U-937 cells. U-937 cells were exposed to TS for 4 h before being labeled with anti-human HSP70 or anti-human Bcl-2; 70% of cells expressed HSP70 after HS (data not shown). Analysis of immunofluorescence, detected with rabbit anti-mouse FITC, was performed by flow cytometry. Values are means ± SE of %HSP70 (n = 8 experiments) and %Bcl-2-(n = 6)-expressing cells. HSP70 was induced with doses between 0.06 and 0.12 puff/ml. Above 0.12 puff/ml, a maximum of 60-70% of HSP70-expressing cells was reached, whereas Bcl-2 was only induced by necrotic concentrations: 0.24-0.96 puff/ml (18-60% of U-937 cells expressing Bcl-2). P values compared TS-treated cells with respective controls.

Bcl-2 expression in U-937 cells exposed to TS. We also investigated the effect of TS on the expression of the oncoprotein Bcl-2, another major antiapoptotic factor. U-937 cells were exposed to TS concentrations ranging from 0.06 to 0.96 puff/ml for 4 or 16 h. Bcl-2 expression levels were then analyzed by flow cytometry. As shown in Fig. 3B, the percentage of cells expressing Bcl-2 after TS exposure for 4 h increased at TS concentrations >= 0.12 puff/ml and reached ~50% of cells for 0.96 puff/ml. Similar levels of induced Bcl-2 were still detectable 16 h after exposure to TS (data not shown). The induction of HSP70 was observed at lower concentrations of TS than the induction of Bcl-2 (compare Fig. 3, A and B).

Effects of HSP Overexpression on TS-Induced Cell Death

Effect of HS pretreatment on TS-induced apoptosis and necrosis. At this stage, it appeared from our data that TS-induced apoptosis occurred despite the parallel induction of antiapoptotic proteins, in particular, HSP70. As an approach to examining whether HSP70 expression could subserve an autoprotective function against TS toxicity, we first analyzed the effects of HS on subsequent TS-induced apoptosis (Fig. 4A) and necrosis (Fig. 4B). U-937 cells were heat shocked at 44°C for 30 min and allowed to recover overnight at 37°C. Indeed, Polla et al. (31) have previously reported that the HS-induced, HSP70-mediated protection against oxidative stress is maximal under these conditions in U-937 cells. The indicated TS concentrations were then applied for 6 h before an assay for cell death was performed. HS pretreatment and subsequent HSP70 overexpression represented a physical stress that potentiated TS-mediated chemical toxicity.


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Fig. 4.   HS has no protective effect on TS-induced apoptosis (A) or necrosis (B) in U-937 cells but instead increases apoptosis and necrosis. U-937 cells were heated at 44°C for 30 min. After overnight recovery, TS concentrations were then applied for 6 h before cell death analysis by flow cytometry. Apoptotic and necrotic cells were detected with annexin V and propidium iodide. Values are means ± SE of %apoptotic or %necrotic cells; n = 4 experiments. Above 0.24 puff/ml, TS toxicity was so high (100% necrotic cells) that no additional toxicity was observed for HS-pretreated cells compared with those without pretreatment. P values compare HS pretreatment with no treatment.

Effect of HSP70 overexpression on TS-induced apoptosis and necrosis. Among the HSPs, HSP70 in particular has been shown to exert antiapoptotic functions (34, 35). To study the direct role of HSP70 in protecting cells from TS-mediated cell injury, we used a rat insulinoma cell line transfected with hsp70 gene for which the overexpression of HSP70 confers resistance against nitric oxide and oxygen species (3). We first confirmed that 4 h of TS exposure induced a concentration-dependent HSP70 overexpression in the rat insulinoma cell line RINm5F similar to that in the U-937 cells and that R70/20 stably expresses a high basal expression (>60%) of HSP70 (data not shown).

Cell death was then analyzed in the RINm5F cells, the R70/20 cells transfected with hsp70 gene, and the control transfected RK/2 cells, which were all exposed to TS for 16 h. For concentrations up to 0.12 puff/ml, TS induced apoptosis (Fig. 5A) and necrosis (Fig. 5B) in the same proportion in both control and hsp70-transfected cells. For higher concentrations (0.24-0.96 puff/ml), hsp70-transfected cells were more resistant to necrosis than RINm5F and RK/2 cells (Fig. 5B). This HSP70-mediated decrease in TS-induced necrosis resulted in a parallel increase in apoptosis (Fig. 5A), the latter effect being a consequence of the former rather than a specific effect of HSP70 in increasing apoptosis.


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Fig. 5.   Effect of HSP70 overexpression in rat insulinoma cells on TS-induced cell death by apoptosis (A) or necrosis (B). RIN5mF (RIN; nontransfected control), R70/20 (transfected with human hsp70 gene), and RK/2 (pZEM-neo transfected control) cells were exposed to TS doses ranging from 0.06 to 0.96 puff/ml for 16 h. Apoptotic and necrotic cells were detected with annexin V and propidium iodide before cytometric analysis. Values are means ± SE of %apoptotic or %necrotic cells; n = 6 for control cells, n = 3 for HSP70-overexpressing cells. Rat cells had same sensitivity to apoptosis and to necrosis as human cells. HSP70-overexpressing cells were less sensitive to necrosis than control RINm5F cells with high concentrations of TS, whereas for the same concentrations, HSP70-overexpressing cells were more sensitive to apoptosis. For TS concentrations ranging from 0.06 to 0.12 puff/ml, no differences were observed in any cell line. P values compare hsp70-transfected cells with respective controls.

The induction of both apoptosis and necrosis in RINm5F cells by TS was again confirmed by electron microscopy (Fig. 6). Control cells (Fig. 6A) displayed distinct morphological features compared with U-937 cells: smoother plasma membrane and an oval, unlobulated nucleus. Low concentration of TS (Fig. 6B) essentially led to perinuclear condensation of chromatin and alterations in cytosolic organelles; notably, mitochondria were not distinguished as clearly as in control cells. Higher concentrations of TS (Fig. 6C) caused typical features of necrosis, i.e., plasma membrane disruption and vacuolization as also observed in U-937 cells (Fig. 2C)


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Fig. 6.   Morphological analysis of RINm5F cells exposed to TS. A: typical aspect of a control RINm5F cell (×9,000). B: in RINm5F cells exposed to low concentrations of TS (0.12 puff/ml) for 16 h, there was chromatin condensation at periphery of nuclear envelope, together with organelle, notably mitochondrial, alterations (×9,000). C: exposure of RINm5F cells to high concentrations of TS (0.48 puff/ml) for 16 h caused typical features of necrosis, such as plasma membrane disintegration and vacuolization (×20,000).

    DISCUSSION
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Introduction
Methods
Results
Discussion
References

We report that TS-exposed mammalian cell lines undergo apoptosis at low concentrations of TS and necrosis at higher concentrations. As early as 4 h after exposure, TS induced an increase in HSP70 for both apoptotic and necrotic TS concentrations but an increase in Bcl-2 only for necrotic TS concentrations. TS- and HS-induced HSPs had no protective effect on either apoptosis or necrosis. In contrast, HSP70-overexpressing cells were protected from TS-induced necrosis. The cells protected against necrosis ended up dying in apoptosis, which could have been interpreted as an HSP70-mediated increase in apoptosis if necrosis had not been examined concurrently.

In both human and rat cell lines, TS concentrations of 0.03-0.12 puff/ml were apoptotic, whereas concentrations of 0.24 puff/ml and above led to necrosis. Our results are consistent with other studies showing that the same biological, chemical, or physical stresses may induce both apoptosis or necrosis depending on the dose and/or the cell type (5, 14). Oxidative stress is known as a broad mediator of apoptosis. TS-mediated induction of apoptosis in rat and human cell lines was, at least in part, due to ROS contained in TS or generated by cells exposed to TS and, in particular, H2O2. Indeed, N-acetyl-L-cysteine and catalase significantly decreased apoptosis in human monocytes exposed to TS, whereas superoxide dismutase had no significant effect (Banzet and Polla, unpublished observation). A direct role of H2O2 in inducing apoptosis has been shown in various cell lines. For example, in the human T-cell line CCRF-CEM, an inverse relationship between extracellular catalase levels and sensitivity to H2O2-induced apoptosis has been reported. Other models of apoptosis, such as TNF-alpha -induced apoptosis, are also mediated by oxidants and thus prevented by various antioxidants.

Because TS induced apoptosis and necrosis in the mammalian cell lines, it was of interest to examine the effects of TS on the expression of antiapoptotic proteins such as HSP70 and Bcl-2. These two proteins do seem to play similar roles in preventing cell death: both protect cells from oxidative damage, decrease lipid peroxidation, inhibit the generation of ROS, and exert effects distal from calcium fluxes (2, 18). Bcl-2 appears to protect cells from apoptosis and necrosis by acting on mitochondrial permeability transition pores, thus preventing the release of proapoptotic factors (such as apoptosis-inducing factor and cytochrome c) (20, 38). A similar mechanism has been proposed for HSP70 (30, 31). In our experiments, Bcl-2 was increased by necrotic TS concentrations only. Despite this overexpression, U-937 cells were not protected from TS-induced cell death, although in other studies, Bcl-2 overexpression was found to protect against H2O2-mediated apoptosis as well as necrosis (14). It is therefore hypothesized in our model that either the increase in Bcl-2 expression level is insufficient to protect cells against necrosis or Bcl-2 has no protective effect toward the specific type of necrosis induced by TS.

If the increase in Bcl-2 was only observed for necrotic concentrations, HSP70, in contrast, was induced by both low and high TS concentrations. HSP70 induction by TS thus appears as a more sensitive marker than Bcl-2 for the stress induced by TS. Polla et al. (31) have previously reported that Bcl-2 is also induced by HS but, again, at lower levels than HSP70. Interestingly, the induction of HSP70 in parallel to apoptosis has been observed in several apoptosis models (6, 15). On the other hand, a number of studies have shown that HSP, and in particular HSP70, can protect cells from ROS such as H2O2 and NO both in vitro and in vivo (30, 3, 29) and from apoptosis induced by a variety of agents (10, 34, 35, 37). Mosser et al. (26) suggested that the HSP70-mediated protective effects against HS-induced apoptosis were associated with reduced cleavage of the death substrate protein poly(ADP-ribose) polymerase. The HSP70-mediated protective effect in NO-mediated toxicity has been shown in rat insulinoma cell lines, and it also involves a downregulation of poly(ADP-ribose) polymerase (4).

However, as indicated by several authors (31, 35), the "protective" effects of HSP70 against apoptosis may actually have deleterious consequences in inflammation or carcinogenesis. To assess whether the increase in HSPs secondary to the stress caused by TS could decrease apoptosis and thus, in the end, potentially enhance the carcinogenic effects of TS, we used two approaches: 1) preexposure to HS, with subsequent overexpression of HSP, under conditions in which we previously demonstrated an efficient, HSP70-mediated protection against ROS; and 2) HSP70 overexpression.

HS pretreatment of U-937 cells did not protect them from TS-induced apoptosis and necrosis; on the contrary, it enhanced both types of cell death. The enhancing effects of HS pretreatment on TS-induced cell death are reminiscent of the potentiation, by hyperthermia, of the cytotoxicity of certain anticancer drugs that occurs via an increase in the rate of uptake of the drug or modulations of intracellular distribution and rate of metabolism (12).

In contrast, HSP70-overexpressing cells were protected from necrosis induced by high concentrations of TS, which is in agreement with a previous study (3) in the same cells showing that HSP70 overexpression protects from cell death caused by NO. In parallel, however, for the same TS concentrations, we observed a higher susceptibility to apoptosis, indicating that, at least with respect to TS exposure, HSP70 has no antiapoptotic effect. These results raise two possibilities, the first one being that HSP70 overexpression would actually enhance apoptosis as it has been described for T-cell receptor- or Fas/Apo-1-mediated apoptosis (23) and for TNF-alpha - and cycloheximide-induced apoptosis (11). In such cases, however, the enhancer effect should also be observed for low concentrations of TS. The second hypothesis is that HSP70 selectively protects cells only from necrotic death without primarily affecting apoptosis. This hypothesis implies that cells that do not die by necrosis will die by apoptosis. Accordingly, HSP70 overexpression should provide an increased resistance to TS toxicity only at necrotic concentrations. The hypothesis on HSP70 protection of necrosis is supported by similar effects of H2O2 in necrotic and apoptotic fibroblasts. In these studies (14), HSP27 inhibits cell death induced by high doses of H2O2 but is inefficient in preventing cell death induced by lower doses. It thus appears that HSPs are indeed able to prevent necrosis induced by TS or H2O2 but are unable to prevent apoptosis. One possible explanation for these differential effects of HSP70 on apoptosis and necrosis is that necrosis is mainly the consequence of cellular damages that can indeed be minimized by overexpression of HSP, whereas low TS or H2O2 concentrations activate an HSP-independent apoptotic program.

Even though the precise determinants of the "decision" of the cell to die by apoptosis or necrosis are still unclear, Richter et al. (33) have proposed that the cellular ATP level is an important parameter of cell death by necrosis or apoptosis. Apoptosis proceeds only when ATP is available, whereas under severe ATP depletion, controlled cell death ceases and cells rapidly die in necrosis (7, 33, 36). To confirm the importance of ATP levels as a switchboard between apoptosis and necrosis in our model, we are currently determining ATP levels in U-937 cells treated with both low and high doses of TS. If ATP levels indeed play a central role in the switchboard between apoptosis or necrosis, one can hypothesize that HSP70 protects from necrosis by rescuing ATP levels in necrotic cells only and that these cells will finally undergo apoptosis. A number of studies (1, 19, 25) that used the constitutive overexpression of hsp70 gene in vitro and in vivo provide direct evidence that HSP70 is involved in the protection of ATP-deprived mammalian cells from injury and death. The observed cytoprotection is associated with an HSP70-mediated preservation of the cytoskeleton, and it will be of interest to correlate the functional effects of HSPs to ultrastructure in our models.

According to our results, HSP70 can inhibit necrosis and subsequently increase apoptosis. If these in vitro studies relate to in vivo events, we can propose that HSP70 is not involved in the carcinogenetic properties of TS. On the other hand, our results of the effect of HSP70 on TS-induced cell death reconcile the apparently contradictory data on the effect of HSPs on apoptosis (increase vs. prevention), i.e., the apoptosis-necrosis paradox (16): cells not dying by necrosis will die by apoptosis, a balance that might generate per se a misleading increase in apoptosis. The apparent apoptosis-enhancing effect of HSP70 is only a logical, although indirect, consequence of necrosis protection. Accordingly, the role of HSP70 in inducing apoptosis proposed by some authors would not relate to a direct effect of HSP70 on apoptosis but would occur as a consequence of HSP70-mediated protection against necrosis. Thus whenever the effects of cell death modulators are investigated, the two types of cell death should be taken into account (14).

    ACKNOWLEDGEMENTS

We are grateful to Prof. David S. Latchman for critical review and stimulating discussions.

    FOOTNOTES

This work was supported by the Société de Secours des Amis des Sciences (M. Vayssier and N. Banzet), the Ciba-Geigy Jubiläums Foundation, and Electricité de France. B. S. Polla was supported by the Institut National de la Santé et de la Recherche Médicale and D. François by the Centre National de la Recherche Scientifique.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: B. S. Polla, Laboratoire de Physiologie Respiratoire, UFR Cochin Port-Royal, 24 rue du Faubourg St. Jacques, 75014 Paris, France.

Received 9 March 1998; accepted in final form 28 May 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Amin, V., D. V. Cumming, and D. S. Latchman. Overexpression of heat shock protein 70 protects neuronal cells against both thermal and ischemia stress but with different efficiency. Neurosci. Lett. 206: 45-48, 1996[Medline].

2.   Barazzone, C., P. Christie, and B. S. Polla. Heat shock proteins in the cell defense mechanisms of the lung. In: Environmental Impact on the Airways: From Injury to Repair, edited by J. Chrétien, D. Dusser, and C. Lenfant. New York: Dekker, 1996, vol. 93, p. 263-284. (Lung Biol. Health Dis. Ser.)

3.   Bellmann, K., M. Jäättelä, D. Wissing, V. Burkart, and H. Kolb. Heat shock protein hsp70 overexpression confers resistance against nitric oxide. FEBS Lett. 391: 185-188, 1996[Medline].

4.   Bellmann, K., A. Wenz, J. Radons, V. Kleemann, and H. Kolb. Heat shock induces resistance in rat pancreatic islet cells against nitric oxide oxygen radicals and streptozotocin toxicity in vitro. J. Clin. Invest. 95: 2840-2845, 1995[Medline].

5.   Buttke, T. M., and P. A. Sandstrom. Oxidative stress as a mediator of apoptosis. Immunol. Today 15: 7-10, 1994[Medline].

6.   Chant, I. D., P. E. Rose, and A. G. Morris. Susceptibility of AML cells to in vitro apoptosis correlates with heat shock protein 70 (hsp70) expression. Br. J. Haematol. 93: 898-902, 1996[Medline].

7.   Chou, C. C., C. Y. Lam, and B. Y. M. Yung. Intracellular ATP is required for actinomycin D-induced apoptosis cell death in HeLa cells. Cancer Lett. 96: 181-187, 1995[Medline].

8.   Cox, G., L. W. Oberly, and G. W. Hunninghake. Manganese superoxide dismutase and heat shock protein 70 are not necessary for suppression of apoptosis in human peripheral blood neutrophils. Am. J. Respir. Cell Mol. Biol. 10: 493-498, 1994[Abstract].

9.   Darzynkiewicz, Z., G. Juan, X. Li, W. Gorczyca, T. Murakami, and F. Taganos. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27: 1-20, 1997[Medline].

10.   Dix, D. J., J. W. Allen, B. W. Collins, C. Mori, N. Nakamura, P. Poorman-Allen, E. H. Goulding, and E. M. Eddy. Targeted gene disruption of hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc. Natl. Acad. Sci. USA 93: 3264-3268, 1996[Abstract/Free Full Text].

11.   Galea-Lauri, J., A. J. Richardson, D. S. Latchman, and D. R. Katz. Increased heat shock protein 90 (hsp90) expression leads to increased apoptosis in the monoblastoid cell line U-937 following induction with TNF-alpha and cycloheximide. A possible role in immunopathology. J. Immunol. 157: 4109-4118, 1996[Abstract].

12.   Galen, W. P. Hyperthermia and chemotherapy. Adv. Exp. Med. Biol. 267: 209-216, 1990[Medline].

13.   Griffith, R. B., and R. Hancock. Simultaneous mainstream-sidestream smoke exposure systems. I. Equipment and procedures. Toxicology 34: 123-138, 1985[Medline].

14.   Guénal, I., C. Sidoti-de Fraisse, S. Gaumer, and B. Mignotte. Bcl-2 and hsp27 act at different levels to suppress programmed cell death. Oncogene 15: 347-360, 1997[Medline].

15.   He, L. S., and M. H. Cox. Variation of heat shock protein 70 through the cell cycle in HL-60 cells and its relationship to apoptosis. Exp. Cell Res. 232: 64-71, 1997[Medline].

16.   Hirsch, T., P. Marchetti, S. A. Susin, B. Dallaporta, N. Zamzami, I. Marzo, M. Geuskens, and G. Kroemer. The apoptotis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15: 1573-1581, 1997[Medline].

17.   Jacquier-Sarlin, M. R., K. Fuller, A. T. Dinh-Xuan, M. J. Richard, and B. S. Polla. Protective effects of hsp70 in inflammation. Experientia 50: 1031-1038, 1994[Medline].

18.   Jager, R., U. Herzer, J. Schenkel, and H. Weiher. Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transgenic mice. Oncogene 15: 1787-1795, 1997[Medline].

19.   Kabakov, A. E., and V. L. Gabai. What are the mechanisms of heat shock protein-mediated cytoprotection under ATP deprivation? In: Heat Shock Proteins and Cytoprotection: ATP-Derived Mammalian Cells. New York: Springer-Verlag, 1997, p. 177-204.

20.   Kluck, R. M., E. Bossy-Wetzel, D. R. Green, and D. D. Newmeyer. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275: 1132-1136, 1997[Abstract/Free Full Text].

21.   Kroemer, G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med. 3: 614-620, 1997[Medline].

22.   Li, W. X., C. H. Chen, C. C. Ling, and G. C. Li. Apoptosis in heat-induced cell killing: the protective role of hsp-70 and the sensitization effect of the c-myc gene. Radiat. Res. 145: 324-330, 1996[Medline].

23.   Liossis, S. N. C., X. Z. Ding, J. G. Kiang, and G. C. Tsokos. Overexpression of the heat shock protein 70 enhances the TCR/CD3- and Fas/Apo-1/CD95-mediated apoptotic cell death in Jurkat T cells. J. Immunol. 158: 5668-5674, 1997[Abstract].

24.   Mailhos, C., M. K. Howard, and D. S. Latchman. Heat shock proteins hsp90 and hsp70 protect neuronal cells from thermal stress but not from programmed cell death. J. Neurochem. 63: 1787-1795, 1994[Medline].

25.   Mestril, R., S. H. Chi, and R. Sayen. Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against stimulated ischemia-induced injury. J. Clin. Invest. 93: 759-767, 1994[Medline].

26.   Mosser, D. D., A. W. Caron, L. Bourget, C. Denislarose, and B. Massie. Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol. Cell. Biol. 17: 5317-5327, 1997[Abstract].

27.   Newcomb, P. A., and P. P. Carbone. The health consequences of smoking. Med. Clin. North Am. 76: 305-331, 1992[Medline].

28.   Pinot, F., A. E. Yaagoubi, P. Christie, A. T. Dinh-Xuan, and B. S. Polla. Induction of stress proteins by tobacco smoke in human monocytes: modulation by antioxydants. Cell Stress Chaperones 2: 156-161, 1997.[Medline]

29.   Plumier, J. C., B. M. Ross, R. W. Curie, C. E. Angelidis, H. Kazlaris, G. Kollias, and G. N. Pagoulatos. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J. Clin. Invest. 95: 1854-1860, 1995[Medline].

30.   Polla, B. S., N. Banzet, J. Dall'Ava, A. P. Arrigo, and A. Vignola. Les mitochondries, carrefour entre vie et mort cellulaire: rôles des HSP et conséquences sur l'inflammation. Med. Sci. 1: 18-25, 1998.

31.   Polla, B. S., S. Kantengwa, D. François, S. Salvioli, C. Franceschi, C. Marsac, and A. Cossarizza. Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc. Natl. Acad. Sci. USA 93: 6458-6463, 1996[Abstract/Free Full Text].

32.   Pryor, W. A., and K. Stone. Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann. NY Acad. Sci. 686: 12-28, 1993[Medline].

33.   Richter, C., M. Schweizer, A. Cossarizza, and C. Franceschi. Control of apoptosis by the cellular ATP level. FEBS Lett. 378: 107-110, 1996[Medline].

34.   Samali, A., and T. G. Cotter. Heat shock proteins increase resistance to apoptosis. Exp. Cell Res. 223: 163-170, 1996[Medline].

35.   Seo, J. S., Y. M. Park, J. I. Kim, E. H. Shim, C. W. Kim, J. J. Jang, S. H. Kim, and W. H. Lee. T cell lymphoma in transgenic mice expressing the human hsp70 gene. Biochem. Biophys. Res. Commun. 218: 582-587, 1996[Medline].

36.   Tsujimoto, Y. Apoptosis and necrosis---intracellular ATP level as a determinant for cell death modes. Cell Death Diff. 4: 429-434, 1997.

37.   Wong, H. R., R. J. Mannix, J. M. Rusnak, A. Boota, H. Zar, S. C. Watkins, J. S. Lazo, and B. R. Pitt. The heat-shock response attenuated lipopolysaccharide-mediated apoptosis in cultured sheep pulmonary artery endothelial cells. Am. J. Respir. Cell Mol. Biol. 15: 745-751, 1996[Abstract].

38.   Yang, J., X. Liu, K. Bhalla, C. N. Kim, A. M. Ibrado, J. Cai, T. Peng, D. P. Jones, and X. Wang. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275: 1129-1132, 1997[Abstract/Free Full Text].


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