Rescue of cells from apoptosis by inhibition of active GSH extrusion

^a Dipartimento di Biologia, Università di Roma Tor Vergata, 00133 Roma, Italy
^b Dipartmento di Scienze Biomediche, Università di Chieti G. D'Annunzio, 66013 Chieti, Italy

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cells induced to apoptosis extrude glutathione in the reduced form concomitantly with (U937 cells) or before (HepG2 cells) the development of apoptosis, much earlier than plasma membrane leakage. Two specific inhibitors of carrier-mediated GSH extrusion, methionine or cystathionine, are able to decrease apoptotic GSH efflux across the intact plasma membrane, demonstrating that in these cell systems GSH extrusion occurs via a specific mechanism. While decreasing GSH efflux, cystathionine or methionine also decrease the extent of apoptosis. They fail to exert anti-apoptotic activity in cells previously deprived of GSH, indicating that the target of the protection is indeed GSH efflux. The cells rescued by methionine or cystathionine remained viable after removal of the apoptogenic inducers and were even able to replicate. This shows that a real rescue to perfect viability and not just a delay of apoptosis is achieved by forcing GSH to stay within the cells during apoptogenic treatment. All this evidence indicates that extrusion of reduced glutathione precedes and is responsible for the irreversible morphofunctional changes of apoptosis, probably by altering the intracellular redox state without intervention of reactive oxygen species, thus giving a rationale for the development of redox-dependent apoptosis under anaerobic conditions.—Ghibelli, L., Fanelli, C., Rotilio, G., Lafavia, E., Coppola, S., Colussi, C., Civitareale, P., Ciriolo, M. R. Rescue of cells from apoptosis by inhibition of active GSH extrusion. FASEB J. 12, 479–486 (1998)

Key Words: GSH efflux • puromycin • methionine • cystathionine

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
APOPTOSIS IS a physiological mode of cell death occurring in processes such as development and differentiation, in tumor cell deletion, and in response to mild damaging stimuli (1–4). This mode of cell death allows the cell to control its own demise. Apoptotic cells have well-defined characteristics: specific DNA digestion, chromatin condensation, and morphological changes such as the actin-dependent phenomena of cytoplasmic blebbing (5) nuclear vesiculation (6). Development of the characteristic apoptotic features depends on the specific proteolytic events that have been found to be involved in the process of apoptosis induced by different stimuli (7). The mechanisms that trigger these events are still far from being clarified: a goal of the current research on apoptosis is to find a point of convergence of the many different apoptogenic stimuli into a main signaling pathway. Many studies indicate that an oxidative mechanism may account for such a central role. Indeed, treatments capable of inducing an oxidative stress are apoptogenic agents. For example, exposure to low doses of H₂O₂ induces apoptosis in a variety of cell types (8, 9); apoptogenic agents such as tumor necrosis factor (10), cycloheximide (11), and natural killer cells (12) are known to elicit oxidative stress. Moreover, the product of the oncogene bcl-2, a protein that is able to block the onset of apoptosis upon many stimuli (13), has been shown to act through a radical-scavenging mechanism (14). This evidence all led to the hypothesis of oxidative stress as a universal trigger for apoptosis, even when the inducing stimuli are apparently unrelated to redox modulation. However, the role of oxidative stress in apoptosis has recently been questioned by reports indicating that apoptosis may occur in anaerobic conditions; i.e., in the absence of reactive oxygen species, which are the most common mediators of oxidative stress (15). Under these conditions, bcl-2 is still able to protect against apoptosis (16).

Glutathione is the most abundant antioxidant in the cell, where it is found predominantly in two redox forms: reduced (GSH)² and oxidized (GSSG). Its protective action is based on oxidation of the thiol group of its cysteine residue with the formation of GSSG, which in turn is catalytically reduced back to the thiol form (GSH) by glutathione reductase (17). Upon oxidative stress, GSSG may either recycle to GSH or exit from the cells, leading to overall glutathione depletion (18). Some cells, mostly hepatocytes, are also able to extrude reduced glutathione through specific carriers, thus maintaining a constant level of GSH in the bloodstream (19). GSH has been shown to prevent apoptosis and maintain viability in cells lacking bcl-2 (20); its concentration decreases upon induction of cells to apoptosis (21), adding support to a causative role of oxidative stress in apoptosis.

We have shown that apoptosis is associated with glutathione depletion in U937 monocytic cells induced to apoptosis by agents that do not imply a direct oxidative stress; glutathione is extruded by the apoptosing cells in the reduced form, before any plasma membrane leakage, indicating that glutathione loss in apoptosis is not a consequence of oxidative stress. We postulated that GSH diminution might be the cause of oxidative stress by altering the reducing power of cells (22).

In this study, we show that apoptotic GSH extrusion occurs through specific carriers and is required for commitment to apoptosis, since we were able to inhibit apoptosis by inhibiting the efflux of GSH in two different cell lines. A rescue to perfect viability was achieved by interfering with GSH efflux from cells during apoptogenic treatment, indicating that in the sequence of events leading to apoptosis, GSH is extruded at a very early step, before any irreversible involution of cellular structure.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell culture
U937 and HepG2 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin, and streptomycin and kept in a controlled-atmosphere (5% CO₂) incubator at 37°C; U937 were split twice a week to maintain them in log phase; HepG2 cells were routinely trypsinized, plated at 7.5 x 10⁵/25 cm² flasks, and used for experiments 3 days after trypsinization. Cell viability for both cell lines was assessed by trypan blue exclusion. All the experiments with U937 cells were performed at a cell density of 7 x 10⁵ cells/ml in complete medium; HepG2 cells were at 2 x 10⁶/25 cm² flasks.

Analysis of apoptosis
Apoptosis in U937 was characterized by DNA fragmentation to give a ladder-like pattern; nuclear fragmentation in several smaller fragments, ranging in number from 2 to more than 20 per cell, was detectable by optical microscopy either on slides of hematoxylin-stained cells or by vital staining with the DNA-specific cell permeable dye Hoechst 33342; cell blebbing was detectable by phase contrast microscopy as the modification of cell shape from nearly round to blackberry-like. HepG2 apoptotic cells were detected with the fluorescence microscope directly on the flasks by analysis of nuclear fragmentation (staining with the DNA-specific cell permeable dye Hoechst 33342) and phosphatidylserine exposure (staining with fluorescinated annexin V).

Preparation and staining of slides
U937 cells (2x10⁵) fixed in 4% paraformaldehyde were loaded onto a gelatinized slide, stained with hematoxylin, and analyzed for direct optical microscopy.

Quantification of apoptosis
The fraction of U937 or HepG2 cells with fragmented nuclei among the total cell population is calculated in the Hoechst 33342 stained cells, counting at least 300 cells in at least 10 random-selected fields as described in ref 9.

Apoptogenic treatments
Treatments with 10 µg/ml puromycin (PMC) and 100 µg/ml etoposide (VP16) were performed in medium supplemented with 10% FCS, for 4 h (U937 cells) or 6–12 h (HepG2), at 37°C; the absence of FCS did not alter the extent or kinetics of puromycin-induced apoptosis.

Recovery
After apoptogenic treatment in the presence or absence of cystathionine or methionine, puromycin was washed out and U937 cells were seeded at the same density (10⁶/ml), with the addition of cystathionine where indicated; at the times indicated, apoptosis was quantitated. For long-term survival, 10⁵ cells/ml were seeded in fresh medium and the viable cells were counted.

Glutathione determination
U937 cells were harvested by centrifugation at 2000 rpm in a refrigerated tabletop centrifuge; HepG2 cells were scraped off before centrifugation. Both samples were then washed with phosphate-buffered saline and resuspended in the same buffer. Cells were then lysated by repeated cycles of freezing and thawing. Proteins were precipitated by adding sodium metaphosphoric acid to a final concentration of 5% (w/v). The clear supernatant obtained after centrifugation at 22,000 g for 15 min was used to measure GSH and GSSG by high-performance liquid chromatography according to Reed et al. (23). In each case, results are expressed as nmol of GSH/mg of protein in the original cell extract. Proteins were determined by processing aliquotes of cell lysates according to the method of Lowry et al. (24). GSH and GSSG measurements of the cell culture medium were determined in serum-free medium as described above after acidification and concentration of the media. No change in the GSH equivalent values were observed in media treated with 5 mM dithiotreitol. Results were expressed as nanomoles of GSH/ml.

Inhibition of GSH efflux
Cells were treated with 1 mM cystathionine or 1 mM methionine; the compounds were added 1 h before the apoptogenic treatment and kept throughout the experiment.

GSH depletion
Glutathione was depleted by 1 h treatment with 2 mM diethylmaleate (DEM), leading to a 90% reduction of intracellular GSH content; DEM was then removed, and GSH synthesis during the apoptogenic treatment was inhibited by 1 mM buthionine sulfoximine (BSO).

Protection from apoptosis
Control cells or cells deprived of GSH by the above-described protocol were treated with either of the following compounds: 10 µg/ml cycloheximide, 1 mM m-iodobenzylguanidine, 5 µg/ml cytochalasin B, 10 mM deoxyglucose, 1 mM cystathionine, or 1 mM methionine. The compounds were added 1 h before the apoptogenic treatment and kept throughout the experiment.

Statistical analysis
Statistical analyses were performed using Student's t test for unpaired data, and P values < 0.05 were considered significant. Data are presented as mean ±SD.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
GSH content and apoptosis of HepG2 cells treated with puromycin
The human hepatoma cells HepG2 undergo apoptosis upon treatment with PMC. Cells begin to acquire apoptotic features such as nuclear fragmentation while still attached; detachment follows, and apoptotic cells float in the culture medium for long periods of time before undergoing secondary necrosis ( Table 1). This allowed us to evaluate separately the intracellular GSH concentration of detached (apoptotic) and attached (healthy + apoptotic) cell populations.

It turned out that detached cells had no detectable GSH or GSSG. Attached cells undergo GSH depletion faster than they acquire apoptotic morphology: in fact, at 6 h, 16% of GSH was lost, but no apoptotic cells were observed; at 12 h, GSH loss was about 85%, but only 48% of cells were apoptotic ( Table 1). These results indicate that glutathione decrease precedes apoptosis in HepG2 cells. Glutathione is lost in the reduced form, which is accumulated in the extracellular culture fluids, whereas no extracellular GSSG was detected nor did the intracellular GSSG/GSH ratio significantly increase in cultures induced to apoptosis ( Table 1).

Effect of cystathionine and methionine on GSH efflux in U937 and HepG2 cells
To understand the mechanism responsible for apoptotic GSH extrusion, we analyzed whether two compounds, cystathionine and methionine, which are known to inhibit specific (synusoidal type) carriers responsible for reduced glutathione efflux from hepathocytes and other cell types (19, 25, 26), are able to inhibit the efflux of reduced glutathione from healthy U937 or HepG2. As indicated in Table 2, both compounds decrease GSH efflux rate, indicating that, not only in hepatoma cells (as established in ref 26), but also in a system of monocytic origin such as the U937 cell line, reduced glutathione is normally shed with a synusoidal-type carrier-mediated mechanism.

We next analyzed whether the specific GSH carriers were also involved in apoptotic GSH extrusion. Thus, U937 and HepG2 cells were treated with 1 mM cystathionine or 1 mM methionine 1 h before and during the apoptogenic treatment. Table 3 shows that inhibitors of GSH carriers do reduce apoptotic GSH efflux induced by puromycin in U937, measured both as the residual intracellular GSH and as accumulation of GSH in the extracellular medium, indicating that it results from a specific extrusion. Similar results were obtained from U937 induced to apoptosis by etoposide and from HepG2 cells treated with PMC (data not shown).

Effect of cystathionine and methionine on apoptosis
To assign a role for GSH efflux in apoptosis, we analyzed the effects that inhibition of GSH efflux may exert on apoptosis. Figure 1A shows the time course of apoptosis induced by puromycin on U937 cells in the presence or absence of cystathionine or methionine. The two compounds have a protective effect throughout the treatment; the fraction of apoptotic cells was repeatedly reduced at all time points in the more than 10 experiments performed, showing that the protective effect is highly significant. The same results were obtained with HepG2 cells, where cystathionine or methionine decreased PMC-induced apoptosis, measured at 12 h of treatment, from 70 ± 9% to 44 ± 5% and 43 ± 8%, respectively ( Fig. 1B). The two compounds also exerted protection ranging between 20 and 36% on etoposide-induced apoptosis (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of cystathionine or methionine on apoptosis and GSH efflux. 1 mM cystathionine (cyst) or methionine (meth) was added to U937 or HepG2 1 h before apoptogenic treatment with 10 µg/ml PMC and maintained throughout the experiment. A) Time course of PMC-induced apoptosis on U937 in the presence or absence of cyst or meth (data are shown as mean±SD, n=10, p<0.01 at 4 h of treatment). B) Viable cells and GSH content at 4 (U937) or 12 h treatment (HepG2).

In Fig. 1B, the effects of cystathionine or methionine on apoptotic GSH efflux and on the extent of apoptosis are compared for U937 and HepG2 cells. For U937, the value of residual GSH concentration in the total cell population matches the value of the residual fraction of viable cells, suggesting that treated, healthy cells have a GSH content identical to that of untreated cells. In HepG2 cells, the value of residual GSH concentration is lower than the value of the residual fraction of viable cells, which indicates a lag between GSH extrusion and the onset of apoptosis. This difference is also observed in time course experiments, which show that in U937, GSH loss is concomitant with apoptosis (22), but in HepG2, GSH loss precedes the loss of healthy morphology ( Table 1).

These results suggest a causative role for GSH efflux in apoptosis, but also raise the possibility that cystathionine and methionine might inhibit the apoptotic process by interfering with an unknown, non-GSH cellular target. In this case, the diminished GSH extrusion could just be a consequence of a lower level of apoptosis.

Analysis of the mechanisms of cystathionine or methionine protection from apoptosis
To investigate the existence of a possible, non-GSH intracellular target of cystathionine or methionine, we analyzed whether the two compounds are still able to offer protection from apoptosis to cells previously deprived of GSH.

Thus, U937 or HepG2 cells were treated with the alkylating agent DEM at the concentration of 2 mM, which under our conditions depleted GSH by 90% in 1 h; DEM was then washed out and 1 mM BSO was added to avoid GSH resynthesis. This treatment by itself was nonapoptogenic on U937 and only slightly apoptogenic on HepG2 cells, and did not affect the kinetics of apoptosis induced by puromycin. On these GSH-depleted U937 ( Fig. 2A) or HepG2 cells ( Fig. 2B), cystathionine or methionine no longer protected from apoptosis, suggesting their effect on apoptosis is indeed mediated by GSH.

View larger version (120K): [in this window] [in a new window]   Figure 2. Analysis of the mechanisms of cystathionine or methionine protection from apoptosis. A) Effect of cystathionine or methionine on apoptosis induced on U937 deprived of GSH. Time course of PMC-induced apoptosis in GSH-deprived U937; cells were treated with 2 mM DEM for 1 h before apoptogenic treatment. DEM was then removed and 1 mM BSO was added to avoid GSH neosynthesis during the apoptogenic treatment (n=5). B) Effect of cystathionine or methionine on apoptosis induced on normal and GSH-deprived HepG2. GSH was depleted as described for U937. Apoptosis was determined at 12 h of treatment. C) Effect of other rescuing compounds on apoptosis induced in U937 deprived of GSH: effect of GSH depletion (see above) on PMC-induced apoptosis in the presence of compounds (citochalasin B, CCB; deoxyglucose, DOG; m-iodobenzylguanidine, mIBG; cycloheximide, CHX), which are known to reduce apoptosis by different mechanisms (see text). Apoptosis was quantified as described at 4 h treatment with PMC. D) Effect of cystathionine or methionine on apoptosis upon inhibition of GSH synthesis. 1 mM cystathionine (cyst) or methionine (meth) was added to U937, as described; where appropriate, 1 mM BSO was added just before apoptogenic treatment with PMC and maintained throughout the experiment (one experiment is reported of 2 performed with similar result). Apoptosis was evaluated at 3 h of PMC treatment.

However, this result may indicate that, alternatively, cells were deeply affected by the GSH deprivation protocol and consequently it may no longer be possible to protect them from apoptosis. Thus, we probed four agents that are able to reduce the extent of apoptosis by four different mechanisms: cycloheximide, which has been shown to reduce puromycin-induced apoptosis on U937 (26); deoxyglucose, which protects from apoptosis by interfering with the glycolytic flux (27); meta-iodobenzylguanidine, which has been shown to protect from apoptosis by inhibiting protein mono-ADP-ribosylation (28); and cytochalasin B, which protects from apoptosis by interfering with cell blebbing (27). As shown in Fig. 2C, all protecting agents were able to reduce puromycin-induced apoptosis on U937 deprived of GSH by the above-mentioned protocol to the same extent they protect cells with normal GSH content. Thus, the inability to protect from apoptosis cells previously deprived of GSH is restricted only to the two agents that interfere with GSH efflux. These experiments clearly demostrate that GSH is the target of cystathionine or methionine protective effects.

It is reported that cystathionine or methionine, in addition to reducing GSH efflux, can increase the rate of GSH synthesis (19, 29). To verify whether the protective anti-apoptotic action of the two compounds was due to this process, we inhibited GSH synthesis with BSO. This treatment, which leads to GSH depletion in the long run (24 h), does not significantly decrease GSH concentration during the first hours. Since we wanted to evaluate the parameter of GSH synthesis rate and not its concentration, apoptosis was induced immediately after BSO addition, before it could lower the cellular GSH concentration. As Fig. 2D shows, cystathionine or methionine were still able to reduce apoptosis when GSH neosynthesis was inhibited by BSO, implying that the mechanism of their protective action was not an increase in the rate of GSH synthesis.

Together, these results indicate that the protective effect of cystathionine or methionine is mediated by inhibition of GSH extrusion.

GSH extrusion occurs before commitment to cell death
The reduction of apoptosis exerted by interfering with GSH efflux might be due to a real rescue of the cells hit by the apoptogenic agent or to a block of the apoptotic process; in the latter, cells might be frozen in a state of pseudo-viability and then proceed into apoptosis when the protecting agent is removed. We followed the fate of cells that had been protected from PMC-induced apoptosis by cystathionine or methionine by testing whether they still need the presence of the `rescuing' compounds in order to remain viable after puromycin has been removed at 4 h of treatment (recovery). We found that after puromycin removal, the cells protected by cystathionine remained viable independent of the presence of the rescuing agent ( Fig. 3A), showing that the protection is no longer needed for maintaining viability. The cells that had been kept viable by cystathionine or methionine continued to remain viable for the ensuing days and were even able to replicate, as indicated in Fig. 3B by the increase in cell number measured at various times after puromycin was washed out in recovery experiments.

View larger version (23K): [in this window] [in a new window]   Figure 3. Fate of U937 cells rescued from apoptosis by cystathionine or methionine. A) Time course of apoptosis upon PMC ± cyst treatment, followed by recovery after removal of PMC (arrow); cyst was removed during recovery in PMC + cyst, but not in PMC + cyst (+cyst rec); one of two similar experiments is shown. B) Cell growth after apoptogenic treatment with PMC ± cyst or met. 105 cells were seeded in fresh medium after the apoptogenic treatment. The number of viable cells was measured at the time points indicated. One of four independent experiments is shown.

Thus, the forced maintainance of GSH inside the cells is itself sufficient to abort the apoptotic program, showing that GSH extrusion occurs before the irreversible commitment to cell death ( Fig. 4).

View larger version (22K): [in this window] [in a new window]   Figure 4. GSH is extruded before irreversible commitment to apoptosis. Induction and execution phases of apoptosis. The dashed vertical line marks the point of irreversible commitment to cell death and the beginning of the involution of cellular structures. GSH extrusion is part of the induction phase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We showed that U937 and HepG2 are protected from apoptosis as a consequence of the inhibition of GSH efflux through specific carriers. The fraction of cells protected from apoptosis by cystathionine or methionine is consistent with the extent of inhibition of GSH depletion at all time points of the many experiments performed with each inducer or cell type. It was not possible to increase the extent of protection, because doses higher than 1 mM were toxic in the systems used. The experiments on HepG2 cells allowed us to establish that glutathione extrusion precedes the morphological changes of apoptosis, because the value of residual GSH concentration is always lower than the value of the residual fraction of viable cells ( Table 1). These results also indicate a lag between GSH extrusion and the onset of apoptosis in HepG2 cells that is not present (or too short to be detected) in U937, where GSH loss and apoptosis appear to be concomitant (22) (however, the recovery experiments allowed us to indirectly determine that GSH extrusion also precedes apoptosis in U937; see below).

The finding that U937 are able to shed GSH may be reminiscent of their monocyte/macrophage origin: it is known that, among the mechanisms of alerting the immune system, macrophages shed cysteine and GSH (31). Cells from the hepatoma line HepG2, which also possess cystathionine/methionine-sensitive GSH carriers, are rescued from puromycin-induced apoptosis by the two compounds. It will be of interest to analyze whether other cell types that are not able to actively shed GSH for a physiological purpose may be rescued from apoptosis by cystathionine or methionine or, instead, whether different types of inhibitors of GSH efflux must be used. As a matter of fact, it has recently been reported that in lymphoid Jurkat cells, GSH is extruded during apoptosis (32); its efflux is unaffected by cystathionine or methionine, but it is sensitive to the canalicular GSH efflux inhibitors bromosulfophtaleins. At variance with our results, the authors also report that the inhibition of GSH efflux does not affect apoptosis. This discrepancy might derive from the different time points considered in the two experimental procedures.

The process of apoptosis may be divided into two distinct phases, induction and execution, whose boundaries signal the irreversible commitment to cell death. We designed an experimental approach that allows one to position an event occurring in apoptosis: if the event under study occurs after commitment, drugs interfering with this event will either delay apoptosis or induce cell death with abnormal characteristics (33). Alternatively, if the event is in the induction phase, its inhibition leads to an abortion of apoptotic signaling, allowing a real rescue of cells. To perform such an analysis, the accumulation of new apoptotic cells must stop upon removal of the apoptogenic agent. Puromycin and etoposide were tested for suitability for this type of analysis by comparing the rate of accumulation of apoptotic cells observed either in the continuous presence of the inducer or after its removal at various times of treatment. We observed that etoposide is not suitable, since after its removal apoptosis continues; instead, apoptosis stops very soon after puromycin removal at any time point (not shown), indicating that it is a good agent to establish whether GSH efflux occurs in induction or execution. Our results show that forced maintainance of GSH inside the cells leads to abortion of the apoptotic signaling, indicating that GSH extrusion occurs before the irreversible commitment to cell death, which is in the induction phase of apoptosis (as a corollary, this implies that GSH loss precedes apoptosis in U937, even though this was not evident from the plain kinetic analysis).

It appears that cells stimulated to undergo apoptosis get rid of their GSH in order to allow apoptosis to take place. GSH loss may be necessary but not sufficient for triggering apoptosis, since chemical glutathione deprivation by BSO and DEM does not induce apoptosis on U937 or HepG2 cells (this study and ref 22). This discrepancy might be alternatively explained by the different modality (i.e., alkilation with DEM or efflux with apoptosis) or rate of GSH depletion (i.e., minutes in apoptosis vs. ~24 h for BSO): cells might slowly `adapt' to a situation of glutathione deprivation by setting up other ways of maintaining a correct redox equilibrium.

Cells deprived of GSH are more prone to undergo oxidative stress because their redox equilibrium is altered and their ability to scavenge or detoxify the various reactive oxygen intermediates that form in the normal cell metabolism is impaired. Active extrusion of GSH could favor the onset of apoptosis by passively allowing oxidative stress to take place. The oxidative enviroment created by GSH loss is independent of the production of reactive oxygen species: this may provide a rationale for apoptosis occurring under low O₂ tension but still requiring a redox modulation. Indeed, it may produce changes in enzymatic activities such as protease activation that are crucial for the triggering of apoptosis. It is known that proteases can be activated through oxidation of a cystein residue (34), and GSH depletion in inflamed pancreatic tissue has been hypothesized to prematurely activate digestive enzymes within pancreatic cells (35). Alternatively, an incorrect redox equilibrium may lead to miscontrol of thiol-dependent ion channels, i.e., the permeability transition pore, a multi-ion mitochondrial thiol-sensitive channel that seems to be responsible for the loss of mitochondrial membrane potential occurring in apoptosis (36), an event that has recently been connected to the apoptotic loss of glutathione (37).

	ACKNOWLEDGMENTS

 
This work was supported in part by CNR special project Progetto Strategico `Ciclo Cellulare e Apoptosi'.

	FOOTNOTES

 
¹ Correspondence: Dipartimento di Biologia, Università di Roma Tor Vergata, via della Ricerca Scientifica, 00133 Roma, Italy. E-mail: ghibelli{at}UTOVRM.IT

² Abbreviations: FCS, fetal calf serum; GSH, reduced glutathione; GSSG, oxidized glutathione; PMC, puromycin; DEM, diethylmaleate; BSO, butionine sulfoximine.

Received for publication August 8, 1997. Accepted for publication December 2, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Wyllie, A. H., Ker, J. F. R., and Currie, A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–273[Medline]
2. Kerr, J. F. R., and Harmon, B. V. (1991) Apoptosis: The Molecular Basis of Cell Death (Tomei, L. D., and Cope, F. O., eds) pp. 5–29, Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y.
3. Wyllie, A. H. (1987) Apoptosis: cell death in tissue regulation. J. Pathol. 153, 313–316[Medline]
4. Buttke, T. M., and Sandstrom, P. A. (1994) Oxidative stress as a mediator of apoptosis. Immunol. Today 15, 7–10[Medline]
5. Cotter, T. G., Lennon, S. V., Glynn, J. M., and Green, D. R. (1992) Microfilament disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Cancer Res. 52, 997–1005[Abstract/Free Full Text]
6. Dini, L., Coppola, S., Ruzittu, M. T., and Ghibelli, L. (1996) Multiple pathways for apoptotic nuclear fragmentation. Exp. Cell Res. 223, 340–347[Medline]
7. Kumar, S. (1995) ICE-like proteases in apoptosis. Trends Biochem. Sci. 20, 198–202[Medline]
8. Lennon, S. V., Martin, S. J., and Cotter, T. G. (1991) Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif. 24, 203–214[Medline]
9. Nosseri, C., Coppola, S., and Ghibelli, L. (1994) Possible involvement of poly(ADP-ribosyl)polymerase in triggering stress-induced apoptosis. Exp. Cell Res. 212, 367–373[Medline]
10. Zeng, L., Xia, Y., Garcia, G. E., Hwang, D., and Wilson, C. B. (1995) Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide. J. Clin. Invest. 95, 1669–1675
11. Zuckerman, S. H., Evans, G. F., and Guturie, L. (1991) Transcriptional and post-transcriptional mechanisms involved in the differential expression of LPS-induced IL-1 and TNF mRNA. Immunol. 73, 460–465[Medline]
12. Malorni, W., D'Ambrosio, A., Rainaldi, G., Rivabene, R., and Viola, M. (1994) Thiol supplier N-acetylcystein enhances conjugate formation between natural killer cells and K562 or U937 targets but increases the lytic function only against the latter. Immunol. Lett. 43, 209–214[Medline]
13. Korsmeyer, S. J. (1992) Bcl-2: a repressor of lymphocyte death. Immunol. Today 14, 131–136
14. Hockenbery, D. M., Oltvai, Z. N., Yin, X. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241–251[Medline]
15. Shimizu, S., Eguchi, Y., Kosaka, H., Kamiike, W., Matsuda, H., and Tsujimoto, Y. (1995) Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature (London) 374, 811–813[Medline]
16. Jacobson, M. D., Raff, M. C. (1995) Programmed cell death and Bcl-2 protection in very low oxygen. Nature (London) 374, 814–816[Medline]
17. Meister, A., and Anderson, M. E. (1983) Glutathione. Annu. Rev. Biochem. 52, 711–760[Medline]
18. Reed, D. J. (1990) Glutathione: toxicological implication. Annu. Rev. Pharmacol. Toxicol. 30, 603–631[Medline]
19. Aw, T. Y., Ooktens, M., and Kaplowitz, N. (1984) Inhibition of glutathione efflux from isolated rat hepatocytes by methionine. J. Biol. Chem. 259, 9355–9358[Abstract/Free Full Text]
20. Kane, D. J., Sarafian, T. A., Anton, R., Hahn, H., Butler Gralla, E., Selverstone Valentine, J., Ord, T., and Bredesen, T. E. (1993) Bcl-2 inhibition of neural death: decreased geration of reactive oxygen species. Science 262, 1274–1277[Abstract/Free Full Text]
21. Slater, A. F. C., Nobel, C. S. I., Maellaro, E., Bustamante, J., Kimland, M., and Orrenius, S. (1995) Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis. Biochem. J. 306, 771–778
22. Ghibelli, L., Coppola, S., Rotilio, G., Lafavia, E., Maresca, V., and Ciriolo, M. R. (1995) Non-oxidative loss of glutathione in apoptosis via GSH extrusion. Biochem. Biophys. Res. Commun. 216, 313–320[Medline]
23. Reed, D. J., Babson, J. R., Beatty, P. W., Brodie, A. E., Ellis, M. W., and Potter, D. W. (1980) High performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulphide and related thiols and disulphides. Anal. Biochem. 106, 55–62[Medline]
24. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275[Free Full Text]
25. Iu, S. C., Sun, W. M., Yi, J., Ookthens, M., Sze, G., and Kaplowitz, N. (1996) Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH mammalian cells. J. Clin. Invest. 97, 1488–1496[Medline]
26. Chow, S. C., Peters, I., and Orrenius, S. (1995) Reevaluation of the role of de novo protein synthesis in rat thymocytes apoptosis. Exp. Cell Res. 216, 149–159[Medline]
27. Ghibelli, L., Noseri, C., Coppola, V., Maresca, V., and Dini, L. (1995) The increase in H₂O₂-induced apoptosis by ADP-ribosylation inhibitors is related to cell blebbing. Exp. Cell Res. 221, 470–477[Medline]
28. Wright, S. C., Wei, Q. S., Kinder, D. H., and Larrick, J. (1996) Biochemical pathways of apoptosis: nicotinamide adenine dinucleotide-deficient cells are resistant to tumor necrosis factor or ultraviolet light activation of the 24-kD apoptotic protease and DNA. J. Exp. Med. 183, 463–471[Abstract/Free Full Text]
29. Kaplowitz, N. (1990) Inhibition of GSH efflux from rat liver by methionine effects of GSH synthesis in cells and perfused organ. Am. J. Physiol. 258, G967-G973
30. Beatty, P. W., and Reed, D. J. (1980) Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes. Arch. Biochem. Biophys. 204, 80–87[Medline]
31. Rouzer, C. A., Scott, W. A., Griffith, O. W., Hamill, A. L., and Cohn, Z. A. (1981) Glutathione metabolism in resting and phagocytizing peritoneal macrophages. J. Biol. Chem. 257, 2002–2008[Abstract/Free Full Text]
32. Van den Dobbelsteen, D., Nobel, S., Schlegel, J., Cotgreave, I. A., Orrenius, S., and Slater, A. (1996) Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-fas/APO1 antibody. J. Biol. Chem. 271, 15420–15427[Abstract/Free Full Text]
33. Ghibelli, L., Maresca, V., Coppola, S., and Gualandi, G. (1995) Protease inhibitors block apoptosis at intermediate stages: a compared analysis of DNA fragmentation and apoptotic nuclear morphology. FEBS Lett. 377, 9–14[Medline]
34. Grierson, A. W., Nicholson, R., Talbot, P., Webster, A., and Kemp, G. (1994) The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis. J. Gen. Virol. 75, 2761–2764[Abstract/Free Full Text]
35. Luthen, R., Niederau, C., and Grendell, J. H. (1995) Intrapancreatic zymogen activation and levels of ATP and glutathione during caerulein pancreatitis in rats. Am. J. Physiol. 268, G592–G604[Abstract/Free Full Text]
36. Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssier, J. L., Petit, P. X., and Kroemer, G. (1995) Reduction in mitochondrial potential constitutes an early and irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661–1672[Abstract/Free Full Text]
37. Macho, A., Hirsch, T., Marzo, I., Marchetti, P., Dallaporta, B., Susi, S. A., Zamzami, N., and Kroemer, G. (1997) Glutathione depletion is an early event and calcium elevation is a late event of thymocyte apoptosis. J. Immunol. 158, 4612–4619[Abstract]