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(The FASEB Journal. 1999;13:69-79.)
© 1999 FASEB

Research Communications

Hydrogen peroxide produced during {gamma}-glutamyl transpeptidase activity is involved in prevention of apoptosis and maintainance of proliferation in U937 cells

Barbara Del Belloa, Aldo Paolicchib, Mario Comportia, Alfonso Pompellaa,1 and Emilia Maellaroa

a Institute of General Pathology, University of Siena Medical School, I-53100 Siena, Italy
b Department of Experimental Biomedicine, University of Pisa Medical School, I-56100 Pisa, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been reported in several cell lines that exposure to low levels of reactive oxygen species can exert a stimulatory effect on their proliferation. We have previously shown that mild oxidative conditions can also counteract apoptotic stimuli. A constitutive cellular production of low levels of superoxide and hydrogen peroxide originates from various sources; among these, {gamma}-glutamyl transpeptidase (GGT), the plasma membrane-bound activity in charge of metabolizing extracellular reduced glutathione, has recently been included. Since the inhibition of GGT is a sufficient stimulus for the induction of apoptosis in selected cell lines, we investigated whether this effect might result from the suppression of the mentioned GGT-dependent prooxidant reactions, on the theory that the latter may represent a basal antiapoptotic and proliferative signal for the cell. Experiments showed that: 1) GGT activity in U937 monoblastoid cells is associated with the production of low levels of hydrogen peroxide, and two independent GGT inhibitors cause a dose-dependent decrease of such GGT-dependent production of H2O2; 2) GGT inhibition with acivicin results in cell growth arrest, and induces cell death and DNA fragmentation with the ladder appearance of apoptosis; 3) treatment of cells with catalase—and even more with Trolox C—is able to decrease their proliferative rate; 4) GGT inhibition (with suppression of H2O2 production) results in a down-regulation of poly(ADP-ribose) polimerase (PARP) activity, which precedes the proteolytic cleavage of PARP molecule, such as that typically induced by caspases. The reported data suggest that the low H2O2 levels originating as a by-product during GGT activity are able to act as sort of a `life signal' in U937 cells, insofar as they can maintain cell proliferation and protect against apoptosis, possibly through an up-regulation of PARP activity.—Del Bello, B., Paolicchi, A., Comporti, M., Pompella, A., Maellaro, E. Hydrogen peroxide produced during {gamma}-glutamyl transpeptidase activity is involved in prevention of apoptosis and maintainance of proliferation in U937 cells. FASEB J. 13, 69–79 (1999)


Key Words: oxidative reactions • acivicin • PARP cleavage • poly(ADP-ribose) polymerase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEVERAL STUDIES HAVE documented the involvement of oxidative stress processes in the determination of apoptosis in various experimental conditions. In fact, many treatments known to induce apoptosis are also able to induce the formation of oxidant mediators, such as reactive oxygen species (hydrogen peroxide in the first place) and nitric oxide. It is also well established that many inhibitors of apoptosis can exert an antioxidant action, either directly or by enhancing cellular antioxidant systems (1). Some degree of oxidative stress has also been repeatedly documented to occur during apoptosis induced by conditions that are not oxidant per se, such as growth factor deprivation (2, 3) and treatment with glucocorticoids (4, 5).

However, based on different lines of research, the involvement of oxidant reactions in the cellular balance between apoptosis and survival appears to be more complex. In fact, evidence has been forwarded that in some cases the exposure of cells to low, nontoxic levels of the reactive oxygen species superoxide and hydrogen peroxide can exert a stimulatory effect on their proliferation, rather than promoting apoptosis or cell necrosis (6, 7). Also, it has recently been reported by our and other laboratories that pretreatment of cells with a mild oxidative stress can result in their protection against apoptogenic stimuli (8–11). For instance, it has been reported that an increased intracellular concentration of superoxide can suppress Fas-mediated apoptosis in sensitive cells (11). Previous studies from our laboratory showed that a short pretreatment with nonlethal concentrations of hydrogen peroxide can prevent apoptosis in thymocytes subsequently exposed to stimuli acting with different mechanisms (8). Thus, the exact role of oxidant reactions in cellular processes involved in apoptosis and proliferation remains as yet undefined.

Another aspect of the picture is highlighted by related studies in which either catalase or superoxide dismutase added exogenously to the growth medium were shown to result in inhibition of cell proliferation, along with the appearance of cell death (12, 13). Although it has not been established yet whether this is a general phenomenon, the observations mentioned led to the speculation that reactive oxygen species, produced basally within the cell, might serve as sort of an autocrine `life signal' capable of maintaining cells in a viable and proliferative state (6).

A basal production of low levels of superoxide and H2O2 exists in normal cells, originating from various sources, e.g., the mitochondria, cytochromes P450 and b5, xantine oxidase, besides the well-known NADPH-oxidase system of phagocytic cells (14). In this respect, recent studies from our laboratory and others have shown that another source of cellular basal production of oxidants is represented by {gamma}-glutamyl transpeptidase (GGT),2 the plasma membrane-bound ectoactivity that, in a number of cell types, is in charge of metabolizing extracellular reduced glutathione (GSH). In fact, it has recently been documented that GGT activity can give rise to redox reactions, due to the interplay of reactive thiol metabolites of GSH (cysteinyl-glycine in the first place) with transition metal ions; these reactions were shown to induce the production of reactive oxygen species and lipid peroxidation (15–18).

It has been shown that the inhibition of GGT is a sufficient stimulus for the induction of apoptosis in T lymphoblastoid cells (19). Hence, bearing in mind the stimulating effects that low levels of oxidants appear to exert in several experimental models, the hypothesis can be formulated that a GGT-dependent generation of oxidant species might represent a basal antiapoptotic and proliferative signal for the cell. The present study was therefore designed to 1) verify whether the apoptotic effect of GGT inhibition is reproducible in a different cell type expressing GGT activity, i.e., the U937 monoblastoid cell line; 2) verify whether the phenomenon is a result of the suppression of the GGT-dependent prooxidant reactions already mentioned; 3) investigate the nature of the prooxidant species produced during GGT activity; and 4) investigate possible relationships between GGT activity, generation of oxidant species and cellular levels of poly(ADP-ribose) polymerase (PARP), an enzyme activity whose involvement in the apoptotic process is well established. The results indicate that hydrogen peroxide is produced during the extracellular GGT-mediated metabolism of GSH, and exerts a protective action against apoptosis and in favor of cell proliferation, possibly by mediating an up-regulation of cellular PARP activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and treatments
Human histiocytic lymphoma U937 cells were cultured in RPMI 1640 medium (Sigma, St. Louis, Mo.) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Sigma), L-glutamine (2 mM), amphotericin B (2.5 mg/l), and gentamycin (50 mg/l), at 37°C, under 95% air:5% CO2 in a humidified atmosphere.

Cells were maintained in logarithmic growth by allowing each subculture to attain a population density of about 1.5–2.0 x 106 cells/ml before reseeding at 2 x 105 cells/ml; for each experiment, cells were seeded at a starting concentration of 5 x 105 cells/ml. Two independent agents were used to inhibit GGT activity: the noncompetitive inhibitor acivicin (AT-125, {alpha}-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, Sigma) (20) and the competitive inhibitor serine/boric acid (5/5 mM) complex. In separate sets of experiments, the following antioxidants were used: superoxide dismutase (50 µg/ml) (Boehringer Mannheim, Mannheim, Germany), catalase (50 µg/ml, dialyzed before using) (Boehringer Mannheim), Trolox C (3 mM, in DMSO 0.3% final concentration) (Fluka, Buchs, Switzerland), desferrioxamine (50 µM) (Ciba-Geigy Basel, Switzerland). Cell membrane integrity was determined by the Trypan blue exclusion test.

In selected experiments, acivicin-treated samples were centrifuged on Ficoll Histopaque (d=1077) (Pharmacia, Uppsala, Sweden). After spinning at 800 x g for 30 min at room temperature, two cell populations were obtained: viable cells (at the interface medium-Ficoll) and apoptotic cells (on the bottom).

Assay of H2O2 release
H2O2 production was measured according to Mohanty et al. (21) by monitoring the horseradish peroxidase (HRP) -catalyzed oxidation of the fluorescent probe N-acetyl-3,7-dihydroxyphenoxazine (A6550), which is nonfluorescent and becomes highly fluorescent only after oxidation by H2O2. At 24 h of incubation, control and acivicin-treated cells were harvested, washed in phosphate-buffered saline (PBS), and resuspended at 5 x 105 cells/ml in Krebs-Ringer phosphate buffer, pH 7.4, containing 145 mM NaCl, 5.7 mM sodium phosphate, 4.9 mM KCl, 0.5 mM CaCl2, 1.2 mM MgSO4, and 1 g/l glucose. Buffered glycyl-glycine and GSH stock solutions were freshly prepared. In the case of serine/boric acid-treated cells, the complex was added to samples just prior to the assay. A6550 (Molecular Probes, Eugene, Oreg.) and HRP (Sigma) were present in all samples at final concentration of 50 µM and 1 U/ml, respectively. After 1 h of incubation at 37°C, the fluorescence of whole samples was measured. The fluorimeter was set at sensitivity 1, and the excitation/emission wavelengths were set at 590/645 nm (slits 4 nm). A standard curve, run from 0.1 to 5 µM H2O2, showing a linear concentration response, was used. The condition of stimulated GGT was achieved by the addition of the acceptor dipeptide glycyl-glycine (10 mM) and GSH (100 µM); for the basal (non-GGT-dependent) H2O2 production, both substrates were omitted. Because of its partial dissociation at neutral pH in the assay buffer, GSH alone is able to oxidize A6550 to a small extent. Therefore, in each experiment the GSH-dependent production of H2O2 was separately determined in the presence of 100 µM GSH, and the value obtained was subtracted from the corresponding GGT-stimulated samples.

Measurement of GGT activity
GGT activity was assayed essentially according to Grisk et al. (22), using {gamma}-glutamyl-p-nitroanilide (Sigma) as substrate of GGT hydrolytic activity and glycyl-glycine (Sigma) as glutamate acceptor for the transpeptidation reaction. In a typical experiment, 1 x 106 cells were incubated for 30 min at 37°C in 0.2 ml of 100 mM Tris-HCl pH 8.0 in the presence of 2.5 mM {gamma}-glutamyl-p-nitroanilide, with or without 20 mM glycyl-glycine. The reaction was stopped by adding 1.8 ml of 1 M sodium acetate (pH 4.0). Cells were then removed by rapid centrifugation and the production of free p-nitroaniline was measured by spectrophotometry at 405 nm. Each experimental value was obtained by subtracting the value of sample minus glycyl-glycine from the value of sample plus glycyl-glycine. GGT activity in control samples at 0 time was 15.4 ± 3.3 mU/mg protein (mean ±SEM of three determinations), where 1 unit is defined as 1 µmol of substrate trasformed/ml/min. The enzyme units were calculated using a molar extinction coefficient of 11,300 for p-nitroaniline formed in the conditions specified above.

Analysis of DNA fragmentation
Colorimetric assay
At the appropriate times, 3 x 106 cells were harvested, washed once with PBS, pH 7.4, and lysed with a lysis buffer (Tris-HCl 5 mM, pH 8.0, 20 mM EDTA, 0.5% Triton X-100). Cell lysates were centrifugated at 20,000 x g for 15 min at 4°C to separate intact chromatin (pellet) from DNA fragments (supernatant). Both pellet and supernatant samples were assayed for DNA content using the diphenylamine reagent (Sigma) (23). Results were expressed as ratio of DNA in the supernatant to total DNA recovered in the pellet plus supernatant.

DNA electrophoresis
DNA from the supernatant, obtained as described above, was used for 1.8% agarose gel electrophoresis to detect nucleosome-sized DNA ladders (24). HaeIII X174RFI DNA fragments were used as molecular weight markers (fragment sizes: 1353 to 72 bp) (USB, Cleveland, Ohio).

GSH determinations
The specific ezymatic method of Tietze (25) was used. For measurements of GSH cellular efflux, U937 cells were incubated (3 h) in RPMI-1640 medium in the presence of 15 or 150 µM acivicin. Cells were then harvested, washed, and incubated in PBS; after 1 h, cells were centrifuged (400 x g) and GSH was determined in supernatants. Intracellular GSH was determined in cell pellets after 24 h of incubation in the various experimental conditions.

Assay of PARP activity
Endogeneous (basal) and stimulated (total) poly(ADP-ribose) polymerase activities were assayed in permeabilized cells, essentially according to Berger et al. (26). At the appropriate times, cells (1x106) were harvested, washed in PBS, and resuspended in 100 µl of ice-cold permeation buffer (Tris-HCl 10 mM buffer, pH 7.8, 1 mM EDTA, 4 mM MgCl2, 3 mM ß-mercaptoethanol, and 0.05% Triton X-100). After 15 min at 4°C, the samples were mixed with 50 µl assay mixture containing 50 mM Tris-HCl buffer, pH 7.8, 4 mM MgCl2, 1 mM ß-mercaptoethanol, 0.5 mg/ml bovine serum albumin, and 30 µM [3H]NAD+ (70 nCi/nmol); for stimulated PARP activity, the assay mixture contained 50 mM Tris-HCl buffer, pH 7.8, 16 mM MgCl2, 1 mM ß-mercaptoethanol, 0.5 mg/ml bovine serum albumin, 30 ng/ml of activating DNA (palindromic octameric deoxynucleotide d-GGAATTCC; Cruachem Ltd, Glasgow, U.K.), and 30 µM [3H]NAD+. The samples were incubated at 30°C for 20 min and the reaction was stopped by the addition of 150 µl of 20% tricholoracetic acid (TCA). The acid-insoluble precipitate was collected on glass microfiber filters (GF/C; Whatman International, Maidstone, England). The filters were washed with 7.5% TCA and cold ethanol, then air-dried before counting (ß-counter, Canberra Packard, Canberra, Australia).

Western blot of PARP
The analysis of PARP protein was performed using the method previously described by Kaufmann et al. (27). Briefly, 2 x 106 cells were spinned and washed in cold PBS for each sample. The supernatant was removed and the pellet resuspended in 100 µl of reducing loading buffer (62.5 mM Tris, pH 6.8; 6 M urea; 10% glycerol; 2% sodium dodecyl sulfate (SDS); 0.003% bromophenol blue; 5% ß-mercaptoethanol). The resuspension was sonicated in ice for 20 s (Vibracell Sonicator, microtips at limit, 40% duty cycle). Samples containing 50 µg of protein were applied to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (5% polyacrilamide for the stacking gel and 8% for the separating gel). After electrophoretic transfer of the separated polypeptides to nitrocellulose membrane (semi-dry Transblot Cell, Pharmacia Biotech), Western blotting was performed using C-2–10, a mouse monoclonal antibody recognizing an epitope located at the carboxyl end of the DNA binding PARP domain (kindly supplied by Dr. G. G. Poirier, Quebec, Canada). A peroxidase-conjugated goat anti-mouse secondary antibody (Boehringer) was used at a dilution of 1:10,000. Signal detection was performed by enhanced chemiluminescence (Boehringer Mannheim).

Other determinations
Protein content was determined by the method of Lowry (28). Statistical significance of data was assessed by the Student's t test for unpaired data. Results are reported as means ± standard error (SEM).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of hydrogen peroxide during GGT activity
Previous studies from our laboratory and others indicated that oxidant processes can occur at the plasma membrane level as a sort of side effect during GGT ecto-enzymatic activity (15–18). To elucidate the nature of GGT-dependent prooxidant effects in U937 cells (exhibiting a GGT activity of approximately 15 mU/mg protein), we performed a direct measurement of reactive oxygen species produced by control and GGT-inhibited cells after 24 h of culture. In control cells incubated in Krebs-Ringer buffer, a basal hydrogen peroxide formation was observed, as measured by the increasing fluorescence of the probe A6550 ( Fig. 1). This value, obtained in the absence of any substrate, represents a basal amount of hydrogen peroxide originating from non-GGT-dependent pathways, allegedly a latent NADPH-oxidase activity of this monoblastoid cell line (29). H2O2 production was much higher (approximately fourfold) under conditions of stimulated GGT, increasing from 1.32 ± 0.05 nmol/106 cells/h in `unstimulated' cells to 5.53 ± 0.35 nmol/106 cells/h in GGT-stimulated cells. Notably, in all instances the production of H2O2 could not take place in the presence of micromolar concentrations of the iron-chelating agent desferrioxamine (not shown), indicating the participation of redox active iron in the reaction; besides, the antioxidants catalase and Trolox C were also able to suppress H2O2 production (not shown).



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  		Figure 1. GGT-dependent hydrogen peroxide production by control and GGT-inhibited U937 cells. Hydrogen peroxide production was determined by fluorescence increase of the probe A6550 (for details, see Materials and Methods). White columns represent the basal H2O2 production (non-GGT-dependent); dark columns represent the total H2O2 production after GGT stimulation. Results are means ±SEM of three experiments. *P < 0.001 as compared to control cells.

While not affecting basal production, GGT inhibitors significantly decreased GGT-dependent H2O2 production ( Fig. 1). In addition, such a decrease paralleled the extent of GGT inhibition obtained with two concentrations of the noncompetitive inhibitor acivicin. As reported in Fig. 2, the continuous exposure of U937 cells to acivicin (for time intervals of up to 2 days) resulted in fact in a dose- and time-dependent inhibition of GGT activity.



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  		Figure 2. Dose- and time-dependent inhibition of GGT enzymatic activity by acivicin in U937 cells. GGT activity was determined spectrophotometrically, as the absorbance of free p-nitroaniline released from the specific substrate {gamma}-glutamyl-p-nitroanilide. Data are means ±SEM of three separate experiments. *P < 0.001 as compared to control activities at the corresponding times.

Effects of GGT inhibition on cell proliferation and survival
To investigate the role played by GGT activity in the cellular balance between apoptosis and survival, cell number was monitored throughout the culture time under conditions of GGT inhibition by acivicin. Both doses of acivicin used caused a severe inhibition of cell growth ( Fig. 3). Since the lower dosage of acivicin was able to block cell proliferation almost completely at 24 h of culture and to inhibit GGT by more than 60% at 4 h (cf. Fig. 2), most subsequent experiments were performed with such a concentration only (15 µM).



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  		Figure 3. Cell proliferation in control and GGT-inhibited U937 cells. Data are means ±SEM of seven experiments. *P < 0.001 as compared to control cell growth at the same culture time.

Concurrently with the growth arrest, in GGT-inhibited cells plasma membrane integrity, as measured by the Trypan blue exclusion test, decreased progressively in time ( Fig. 4A); in addition, most Trypan blue-positive cells appeared reduced in size (not shown). The cell death observed was the consequence of an apoptotic process, as evaluated by determining the percentage of soluble DNA ( Fig. 4B). In fact, the degree of apoptosis was negligible in control, proliferating cells for up to 3 days of culture, whereas in GGT-inhibited cells, soluble DNA was significantly increased after 48 h of acivicin treatment.



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  		Figure 4. Time course of cell death and DNA fragmentation in control and GGT-inhibited U937 cells. A) Plasma membrane integrity, measured as % of Trypan blue-positive cells; B) extent of apoptosis, measured as % of DNA fragmentation. Results are means ±SEM of four separate experiments. *P < 0.01, **P < 0.002, and ***P < 0.001, as compared to the control values at the corresponding culture times.

As shown in Fig. 5, DNA analysis performed by standard agarose gel electrophoresis confirmed the occurrence of a bona fide apoptotic process, resulting in the appearance of typical oligonucleosome-sized DNA fragments in acivicin-treated cells at 48 and 72 h of culture ( Fig. 5A). After Ficoll density-gradient centrifugation of acivicin-treated samples ( Fig. 5B), two subpopulations were obtained: F1 (low-density fraction, enriched in viable cells), showing a light intensity of the oligonucleosomal bands; and F2 (high-density fraction, enriched in apoptotic cells), in which DNA analysis showed a very strong ladder appearance.



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  		Figure 5. DNA agarose gel electrophoresis in control and GGT-inhibited U937 cells. A): Lane 1, DNA standard; lanes 2 and 4, control cells; lanes 3 and 5, acivicin-treated (15 µM). B) Analysis of two distinct cellular fractions, see Materials and Methods; lane 1, DNA standard; lane 2, untreated control (viable cells); lane 3, acivicin-treated, F1 fraction (viable cells); lane 4, acivicin-treated F2 fraction (dead cells). A typical experiment of three is reported; repeated experiments gave fully comparable results.

Effects of GGT inhibition on glutathione efflux and cellular content
As mentioned above, a continuous efflux of GSH has been documented in various cell types (30). Even in the absence of exogenously added GSH, cellular GGT is continuously active in these cell types insofar as it functions as a means for the reuptake of GSH that cells would otherwise lose in the interstitium. Data reported in Table 1 show that this is also the case for U937 cells used in the present study: in fact, the inhibition of GGT by acivicin treatment resulted in the accumulation of GSH in the extracellular medium, indicating that a significant GSH efflux is indeed present in U937 cells. Nevertheless, no decrease of intracellular GSH content was observed in the presence of acivicin under our experimental conditions for up to 48 h of incubation ( Table 2).


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  		Table 1. GSH net efflux in control and acivicin-treated U937 cells1tfa


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  		Table 2. Intracellular GSH in control and acivicin-treated U937 cells at 24 and 48 h of incubation2tfa

Effects of antioxidant treatments on cell proliferation rate
It has been repeatedly demonstrated that low levels of oxidants can play a role in maintaining cell proliferation in various cell models (6). To evaluate whether the growth arrest observed after GGT inhibition could depend on the suppression of a mild GGT-dependent oxidative stress (production of H2O2), U937 cells were exposed to antioxidant treatments and the cell number was monitored over time ( Fig. 6). Superoxide dismutase did not produce appreciable effects, whereas catalase (and Trolox C even more) inhibited cell proliferation at 48–72 h of culture; in any case, cell viability was not significantly affected by either treatment (data not shown).



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  		Figure 6. Effect of different antioxidants on U937 cell proliferation. The antioxidants were added at the beginning of the experiments and maintained throughout the incubation time. *P < 0.05, **P < 0.005, and ***P < 0.001, compared to the corresponding control values.

Effects of GGT inhibition on PARP activity and molecular integrity
Previous studies showed that mild oxidant stimuli are able to prevent apoptosis through an early up-regulation of PARP (5). Against this background, we have examined PARP activity in basal conditions and in the presence of palindromic DNA sequences (the latter condition giving a measure of the stimulated enzyme activity). As reported in Fig. 7, both basal and stimulated PARP activities were significantly decreased in GGT-inhibited cells as compared to control cells, during all culture times studied.



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  		Figure 7. Time course of PARP activity in control and GGT-inhibited U937 cells. PARP activity was measured as [3H]NAD+ incorporation into proteins of permeabilized cells; see Materials and Methodss for details of basal (A) and stimulated (B) activity. Data shown are means ±SEM of three or four experiments. *P < 0.02, **P < 0.005, and ***P < 0.001, as compared to control activities at the corresponding times; aP <0.01 and bP <0.001, as compared to 0 time value.

In the subpopulation selectively enriched in frankly apoptotic cells obtained after density gradient separation, as shown above, PARP activity was dramatically decreased ( Fig. 8). However, this drop did not account for the lowered activity observed in the whole sample of acivicin-treated cells. In fact, a remarkable decrease of PARP activity was also detectable in the subpopulation of still viable cells (i.e., still negative for Trypan blue uptake, showing a minor DNA ladder, and still preserving a normal density) (cf. Figs. 5 and 8).



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  		Figure 8. Decrease of PARP activity in dead cells as well as in still viable cells after GGT inhibition with acivicin. After 72 h of incubation, acivicin-treated cells were separated in two distinct subfractions, as described in Fig. 5. Both basal (A) and stimulated PARP activity (B) was measured. Data shown are means ±SEM of four separate experiments. *P < 0.01, **P < 0.005, and ***P < 0.001, as compared to the corresponding control activity.

To investigate whether the observed decrease in PARP activity was the result of a proteolytic cleavage of PARP protein, we examined PARP integrity in control and GGT-inhibited cells by means of immunoblot, using anti-PARP monoclonal antibodies. As shown in Fig. 9, at 48 and 72 h of culture a single major band of 116 kDa was detectable in control cells, corresponding to intact PARP. In contrast, an additional 89 kDa band was evident in acivicine-treated cells, corresponding to the fragment that typically results from the break of PARP molecule between its DNA binding domain and the rest of the molecule, including automodification and catalytic domains (31).



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  		Figure 9. Cleavage of PARP protein induced in U937 cells by GGT inhibition with acivicin. Western blot was performed using the specific monoclonal antibody C-2–10. Lanes 1 and 3, controls; lanes 2 and 4, acivicin-treated (15 µM) cells. A typical experiment of three is reported; repeated experiments gave fully comparable results.

As described above, treatment of U937 cells with the antioxidant Trolox C also resulted in a significant inhibition of cell growth, even if in absence of signs of apoptosis. Since the cell growth arrest after the inhibition of GGT activity by acivicin was accompained by a decrease in PARP activity, experiments were performed in order to check whether also Trolox C was in some way also interfering with PARP activity. Indeed, exposure of cells to 3 mM Trolox C for 72 h resulted in an approximately 50% decrease in PARP total activity, as reported in Table 3.


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  		Table 3. PARP activity in control and Trolox C-treated U937 cells3tfa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The apoptosis induced by acivicin in different cell lines has been tentatively explained by the fact that inhibition of GGT, by impairing the uptake of {gamma}-glutamyl residues into the cell, would decrease the cellular de novo synthesis of GSH, thus depleting one of the major cellular antioxidants (19). Indeed, depletion of GSH has been observed repeatedly during induction of apoptosis by several different agents (5, 32, 33). However, a peculiar aspect of the experimental model described in the present sudy lies in the fact that no decrease of total intracellular GSH content was observed in up to 48 h of incubation ( Table 1). This is probably due to a compensatory increase in the GSH synthetic pathways set into motion by the inhibition of GGT, as reported by others in different cell systems (34, 35). On the other hand, it has been documented that the activity of GGT at the surface of plasma membrane is accompained by the occurrence of redox reactions, leading to the formation of oxidizing species (15–18). In fact, due to the GGT-mediated cleavage of the {gamma}-glutamyl moiety from GSH, cysteinyl-glycine (GC-SH) is originated, i.e., a thiol provided with much higher reactivity as compared to the parent compound. Since the pKa of GC-SH is about 6.4 and that of GSH is higher than 8.5 (36), at neutral pH GC-SH will mostly be in its thiolate anion dissociated form; besides, GC-SH also lacks the {alpha}-carboxyl group of glutamate, whose presence in GSH has been shown to prevent the interactions of cysteine -SH group with transition metal ions (37). GC-S- thiolate anion will thus be able to start redox interactions with, for example, iron ions present in the medium, leading to the formation of thiyl radicals and reactive oxygen species according to the following scheme:




Thus, the formation of H2O2 as a by-product during GGT activity would depend on the availability of iron ions. Actually, no exogenous source of iron was included in the experimental systems used in the present study. However, it is well established that standard laboratory buffers and cell culture media do contain trace amounts of contaminant iron (14); in fact, when the iron chelator desferrioxamine was included in the assay mixture, no GGT-dependent production of H2O2 could be observed.

A GGT-dependent production of hydrogen peroxide by U937 cells was observed in the presence of exogenous GSH plus the acceptor dipeptide glycyl-glycine (= stimulation of GGT activity) ( Fig. 1). On the other hand, in GGT-inhibited cells (in which proliferation was also inhibited), H2O2 formation was significantly decreased ( Fig. 1). On the basis of these observations, it can be speculated that GGT ectoenzymatic activity—besides its well-known function in the salvage pathway of extracellular GSH—might be a factor as well in stimulation and/or maintainance of U937 cell proliferation through the production of low amounts of hydrogen peroxide. GGT inhibition would actually remove this mild, `beneficial' oxidant stimulus somehow involved in promotion of cell proliferation. Such a speculation is strengthened by the observation that U937 cell proliferation was also inhibited when extracellular H2O2 was removed with catalase, and even more so when the antioxidant Trolox C was included in the culture medium (cf. Fig. 6). Accordingly, Trolox C was in fact able to prevent GGT-mediated H2O2 formation (data not shown). Trolox C is an efficient reductant for several free radicals including, for example, O2O2{bullet}- (38), and it is therefore likely that it can interfere with GGT-mediated prooxidant reactions upstream of the H2O2 production.

Proliferation and apoptosis studies were performed in RPMI-1640 medium, with no exogenously added GSH or glycyl-glycine. However, as indicated by the GGT inhibition studies reported in Table 1, a continuous efflux of GSH from U937 cells occurs that continuously supplies extracellular GSH for GGT activity. Thus, the latter is indeed continuously operative, effecting a sort of GSH cycling across the plasma membrane. Furthermore, RPMI-1640 medium contains a mixture of amino acids that are well suited to perform as {gamma}-glutamyl acceptors. On the other hand, RPMI-1640 medium also contains 10% FCS, which is known to be a good scavenger of H2O2 due for instance to the presence of GSH peroxidase (39), and could thus prevent the accumulation of H2O2. Nevertheless, it is conceivable that significant concentrations of H2O2 could be achieved in the immediate surroundings of the sites where H2O2 is generated.

The results reported are consistent with considerable evidence in the literature suggesting that redox reactions occurring at the plasma membrane level can play regulatory roles in signal transduction pathways. Reactive oxygen species are now recognized as true signal molecules under subtoxic conditions, i.e., molecules able to transfer information from outside of the cell to inside, thus modulating diverse biological activities, such as gene expression, and the balance between cell growth and death (6, 7). Accordingly, recent reports point to the proapoptotic role played under some circumstances by antioxidants, e.g., via the activation of p53-dependent pathways (40, 41).

Growth arrest effected by GGT inhibition in U937 cells was accompanied by the occurrence of apoptotic cell death, as previously reported in other cell lines (19). It is now realized that the proteolytic cleavage of key substrates represents an important feature of the apoptotic machinery, which, however, is still poorly understood in many of its underlying biochemical mechanisms. Several IL-1ß-converting enzyme-like cysteine-proteases (recently termed caspases) have been identified, with high degrees of homology in phylogenetically different cells. Once activated, presumably through a hierarchical cascade of proteolysis, these enzymes become involved in the execution of the apoptotic program and finally cleave downstream proteins that are essential for cellular repair (42). PARP was the first specific substrate of a subgroup of caspases (including Yama/apopain/CPP-32) to be identified in apoptosis, and its cleavage is considered to be essential for the completion of the process.

Our present findings demonstrate that in apoptosis of U937 cells induced by GGT inhibition, a 89 kDa fragment of PARP molecule is originated, as typically results from the activation of the above-mentioned subgroup of caspases (42). With respect to the enzymatic activity of PARP (for reviews of this enzyme, see refs. 43 and 44), few (if any) studies are available in the literature analyzing PARP structure during apoptosis along with possible modifications of PARP catalytic properties, yet the latter would be of great interest in order to understand possible mechanistical roles of this enzyme in the apoptotic process. We show here that in acivicin-treated cells, both basal and stimulated PARP activities were significantly decreased in the whole population compared to control cells; in addition, the decrease was even more evident in a subpopulation enriched in apoptotic cells and was also present in a subpopulation of cells still viable, but presumably committed to apoptosis.

The proteolytic loss of the PARP DNA binding domain observed in apoptotic cells might account for the lower responsiveness of the enzyme to exogenously applied DNA fragments (= stimulated activity). No detectable PARP cleavage was revealed in GGT-inhibited, nonapoptotic cells (data not shown); nevertheless, as already mentioned, PARP activity (both basal and stimulated) was markedly decreased in these cells compared to controls. This suggests that such a decrease in GGT-inhibited cells must be accounted for by some modification of PARP other than its proteolytic cleavage. Since no decrease in PARP protein (as revealed by immunoblots) appeared to occur in GGT-inhibited cells compared to controls, the observed loss of enzymatic activity could in principle be ascribed to posttranslational modifications of PARP (e.g., changes in redox status, phosphorylation, autoribosylation), possibly leading to an impaired substrate affinity.

Another aspect that warrants serious consideration is the progressive increase in time of both basal and stimulated PARP activities observed in control, proliferating cells. A higher level of PARP activity has been described in nuclei of dividing cells (of normal or neoplastic phenotype) compared to the corresponding resting cells (45–47). In our experiments, the treatment of U937 cells with Trolox C consistently induced a marked inhibition of PARP activity, along with depression of the proliferation rate (cf. Fig. 6and Table 3). It was also shown in cultured rat hepatocytes that PARP and GGT activities are related to each other in dependence of cell cycle, thus establishing a link between the two enzymes and cell proliferation (48). GGT expression is an important phenotypic change associated with neoplastic transformation; in fact, it has been long known as a marker of neoplastic progression in several experimental models of chemical carcinogenesis, and occurs at significant levels in a number of human malignant neoplasms and their metastases (49–51). Since increased PARP levels have also been related to tumor cell growth (44), it could be speculated that both PARP and GGT activities may be coordinately involved in some aspects of tumor progression.

Being a glutamine analog, acivicin is known to inhibit glutamine utilization by amidotransferases in nucleotide biosynthesis (52); on this basis, acivicin and other glutamine analogs (e.g., azaserine, 6-diazo-5-oxo-L-norleucine) have been described as antiproliferative agents. Nevertheless, the antiproliferative action of acivicin could not be reverted by the administration of preformed nucleotides, suggesting a minor role for amidotransferases in the maintenance of cell proliferation (53). On the other hand, the glutamine analogs mentioned are also potent inhibitors of GGT activity (54). Against this background, the inhibition of GGT-dependent H2O2 production could be a major aspect of the biological activity of acivicin and other glutamine analogs. In this perspective, the observation of varying levels of GGT activity in different tumors could help explain the inconstant therapeutic outcome of glutamine analogs when used as antineoplastic drugs (52).

In conclusion, data reported in the present study can be summarized according to the chart reported in Fig. 10. H2O2 formed during the GGT ectoenzymatic activity [1] appears to stimulate and/or maintain U937 cell proliferation [2], which is inhibitable by antioxidant treatments [3]. GGT inhibition effected by acivicin [4] (and by complex serine/boric acid) can decrease H2O2 formation and (as a consequence?) cell proliferation; concomitantly, GGT inhibition is followed by a decrease in PARP activity and by the onset of apoptosis [5]. The removal of a `physiological' production of extracellular H2O2 by GGT inhibition could determine the observed decrease of PARP activity through its cleavage [6]. Between the inhibition of H2O2 formation at the plasma membrane level and modifications of PARP activity in the cell nucleus, a number of biochemical events can be hypothesized in an attempt to explain the temporal sequence and intracellular signaling pathways involved. A possibility is that the more reduced status of membrane protein thiols, as occurs after GGT inhibition (A. Paolicchi, E. Maellaro, and A. Pompella, unpublished observations), can modulate the signaling function of critical proteins involved in the apoptotic process [7]. Studies are in progress in our laboratories to elucidate further aspects of this apparently complex picture.



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  		Figure 10. Outline of possible interactions between GGT activity, production of hydrogen peroxide, cell proliferation, PARP activity, and apoptosis.


   ACKNOWLEDGMENTS
 
This work was supported by the Associazione Italiana per la Ricerca sul Cancro and by MURST-Cofinanziamento '98 Funds. The financial support of the Association for International Cancer Research is also gratefully acknowledged.


   FOOTNOTES
 
1 Correspondence: Institute of General Pathology, Via Aldo Moro, I-53100 Siena, Italy. E-mail: apompella{at}unisi.it

2 Abbreviations: FCS, fetal calf serum; GC-SH, cysteinyl-glycine; GGT, {gamma}-glutamyl transpeptidase; GSH, glutathione; HRP, horseradish peroxidase; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCA, tricholoracetic acid.

Received for publication July 14, 1998. Revision received September 18, 1998.
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