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(The FASEB Journal. 2001;15:1927-1940.)
© 2001 FASEB

Efficiency of homocysteine plus copper in inducing apoptosis is inversely proportional to {gamma}-glutamyl transpeptidase activity

GINETTE BESSEDE, CAROLE MIGUET, PHILIPPE GAMBERT, DOMINIQUE NEEL and GERARD LIZARD1

Inserm U498 (Métabolisme des lipoprotéines humaines et interactions vasculaires), CHU/Hôpital du Bocage, 21034 Dijon Cedex, France

1Correspondence: CHU/Hôpital du Bocage, Inserm U498, Laboratoire de Biochimie Médicale, BP 1542, 2 Bd Maréchal de Lattre de Tassigny, 21034 Dijon Cedex, France. E-mail: Gerard.Lizard{at}u-bourgogne.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperhomocysteinemia represents an independent risk factor for atherosclerosis, but the mechanisms leading to cellular dysfunctions remain unknown. Using ECV304 cells, we found that homocysteine (Hcy) plus copper (Cu2+) induced cytotoxic effects: loss of cell adhesion, increased permeability to PI, and the occurrence of morphologically apoptotic cells. This form of apoptosis, inhibited by Z-VAD-fmk, was associated with a loss of mitochondrial potential, a cytosolic release of cytochrome c, activation of caspase-3, degradation of poly(ADP-ribose)polymerase, and internucleosomal DNA fragmentation. However, the ability of Hcy plus Cu2+ to induce apoptosis decreased when the pretreatment culture time increased. As a positive correlation was found between the length of time of culture before treatment and the enhancement of {gamma}-glutamyl transpeptidase ({gamma}-GT) activity, we asked whether {gamma}-GT was involved in the control of Hcy plus Cu2+-induced apoptosis. Therefore, ECV304 cells were treated with either acivicin or dexamethasone, inhibiting and stimulating {gamma}-GT, respectively. In ECV304 cells and human umbilical venous endothelial cells, acivicin favored Hcy plus Cu2+-induced apoptosis whereas dexamethasone counteracted the apoptotic process. As acivicin and dexamethasone were also capable of modulating cell death in ECV304 cells treated with antitumoral drugs, our data emphasize that the involvement of {gamma}-GT in the control of apoptosis is not restricted to Hcy but also concerns other chemical compounds.—Bessede, G., Miguet, C., Gambert, P., Neel, D., Lizard, G. Efficiency of homocysteine plus copper in inducing apoptosis is inversely proportional to {gamma}-glutamyl transpeptidase activity.


Key Words: HUVECs • ECV304 cells • Hcy • {gamma}-GT


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ATHEROSCLEROSIS IS A complex phenomenon (1 , 2) and hyperhomocysteinemia is an important risk factor for atherosclerotic disease in coronary, cerebral, and peripheral vessels (3 , 4) . Homocysteine (Hcy), an intermediate compound formed during metabolism of methionine (5) , normally is present in the plasma of healthy people at ~10 µmol/l (6 , 7) but its plasma concentration can reach up to 500 µmol/l and higher in patients suffering from hyperhomocysteinemia due to either homozygous deficiency of cystathionine ß-synthase (6 , 8) or mutations in the genes for 5, 10-methylene tetrahydrofolate reductase and/or for methionine synthase (9 , 10) . Other factors associated with increased plasma levels of Hcy include increased age, deficiency in folic acid, vitamin B6 and B12, impaired renal function, and several medications (4) . Patients with an inborn or acquired defect of Hcy metabolism leading to hyperhomocysteinemia develop atherosclerosis (11) , vascular occlusions (12) , and thromboembolic events (13) at an early age. Some clinical studies have also reported the involvement of mild homocysteinemia (15 to 35 µmol/l) in the development of atherosclerotic lesions (14) . Moreover, Hcy has been shown to induce inhibition of endothelium-dependent relaxation from rat aorta (15) as well as arteriosclerosis-like alterations on baboons (16) , rhesus monkeys (17) , pigs (18) , and rats (19) . Taken together, these different observations have contributed in establishing that hyperhomocysteinemia constitutes an independent risk factor for atherosclerosis (20) , but the underlying pathophysiological mechanisms leading to vascular injuries remain to be elucidated.

Nowadays there is evidence for copper (Cu2+) -dependent toxic effects of Hcy on a wide number of cells including those of the vascular wall (endothelial cells and smooth muscle cells). On HeLa and ECV304 cells, the effects of the combined use of Hcy and Cu2+ decreased the intracellular concentration of glutathione and increased the release of glutathione into the medium (21) . On vascular smooth muscle cells isolated from rat thoracic aorta, Hcy lowered the glutathione peroxidase activity, enhanced the superoxide dismutase activity, and stimulated the production of H2O2 (22) . In the presence of Cu2+, Hcy can also trigger generation of reactive oxygen species by human peripheral blood mononuclear cells (23) . Numerous cellular dysfunctions were observed in Hcy-treated endothelial cells from various animal species, including humans. Thus, Hcy reduces antithrombin III binding capacity of cell surface heparan sulfate (24) as well as thrombomodulin surface expression (25) and also increases factor V activity (26) . These observations may account, at least in part, for the increase incidence of thrombosis in patient with hyperhomocystinemia (4) . In addition, exposure of cultured endothelial cells to Hcy reduces cell growth (27) , increases the amount of telomere length lost per population doubling, and increases the expression of cell surface molecules linked to vascular disease such as intracellular adhesion molecule 1 and plasminogen activator inhibitor 1 (28) .

Since dysfunctions of the endothelium seem to play important roles in atherosclerosis resulting from hyperhomocysteinemia (29) , we attempted to characterize the mode of cell death (apoptosis vs. necrosis) (30) induced by the combined use of Hcy and Cu2+ in ECV304 cells, which present certain characteristics of endothelial cells (31 32 33 34) , as well as in human umbilical venous endothelial cells (HUVECs). As the cytotoxicity of Hcy plus Cu2+ is probably due at least in part to the production of hydrogen peroxide (22 , 23 , 35) , capable of modifying the redox status (21 , 36) and catalyzing DNA degradation (37) , we tried to link these characteristics with the process of cell death. Since tripeptide thiol glutathione plays a pivotal role in the control of cellular functions (38 , 39) and cell death induced by numerous agents (40) , we examined the effects of Hcy plus Cu2+ on the ectoenzyme {gamma}-glutamyl transpeptidase ({gamma}-GT), which catalyzes the first step in the extracellular transpeptidation of glutathione via the {gamma}-glutamyl cycle into amino acid intermediates (39 , 41) . Indeed, in different tumoral cell lines, the ability of various drugs to induce apoptosis has been shown to depend on the level and activity of {gamma}-GT; thus, cells with high {gamma}-GT levels and activity were often resistant to antitumoral drugs (42 , 43) .

In the present study, we demonstrate with ECV304 cells and HUVECs that Hcy plus Cu2+ induces an apoptotic process. This mode of cell death is characterized by inhibition of cell growth, a loss of cell adhesion, increased permeability to propidium iodide (PI), a drop in transmembrane mitochondrial potential ({Delta}{Psi}m), a cytosolic release of cytochrome c, activation of caspase-3, degradation of poly(ADP-ribose)polymerase, an internucleosomal DNA degradation, and the occurrence of cells with fragmented and or condensed nuclei characteristic of apoptotically dying cells (44) . This form of apoptosis was inhibited by N-benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone (Z-VAD-fmk), which is a broad-spectrum, cell-permeable caspase inhibitor (45) . In addition, we show that the potency of Hcy plus Cu2+ to trigger apoptosis depended on the period of culture (3, 4, or 5 days) before treatment. Thus, the highest efficiency of Hcy plus Cu2+ in triggering apoptosis was observed when the cells had been cultured for only 3 days. Since {gamma}-GT activity was found to increase with the time of culture, we have attempted to demonstrate that this parameter was involved in the control of cell death. This involvement of {gamma}-GT in Hcy plus Cu2+-induced apoptosis was underscored by cotreatment of the cells with acivicin and dexamethasone. Under these conditions, acivicin, which strongly inhibits {gamma}-GT activity (46) , indeed favored Hcy plus Cu2+-induced apoptosis whereas dexamethasone, which stimulates {gamma}-GT activity (47) , counteracted the apoptotic process. Moreover, the capability of acivicin and dexamethasone to favor and inhibit apoptosis, respectively, was also observed when ECV304 cells were treated with antitumoral drugs such as daunorubicin, etoposide (VP-16), vinblastine, and cytosine ß-D-arabinofuranoside (Ara-C). Therefore, the key role of {gamma}-GT in the control of apoptosis is not restricted to Hcy plus Cu2+-induced cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
ECV304 cells initially described as endothelial cells isolated from umbilical cord vein (31) were obtained from the American Type Culture Collection (Manassas, VA). Whereas the origin of ECV304 cells is now controversial (48) , these cells nevertheless have some endothelial characteristics (32 33 34) . Moreover, when reverse transcription polymerase chain reaction (RT-PCR) was performed as described (49) with the respective forward (F-) and reverse (R-) primers specific for human endothelial nitrous oxide synthase (ecNOS) (ecNOS primers; F-[5'-AAG CCG CAT ACG CAC CCA GAG-3'], R- [5'-TGG GGT ACC GCT GCT GGG AGG-3'], product size 346 bp), ecNOS mRNA was detected in ECV304 cells. In the present work, ECV304 cells were seeded at 13 x 103 cells per cm2 in 75 cm2 tissue culture flasks (Falcon/Becton-Dickinson, Plymouth, UK) containing 20 ml of culture medium [medium 199 with Earle’s salts, 2.3 g/l NaHCO3, amino acids, and Glutamax (Life Technologies, Inc., Eragny, France), antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin; Life Technologies), 10% (v/v) heat-inactivated fetal calf serum (Boehringer Ingelheim, Gagny, France)] and passaged once a week.

HUVECs were isolated from umbilical cord veins as described (50) and used at the first passage. They were seeded at 30 x 103 cells per cm2 in 75 cm2 tissue culture flasks (Falcon/Becton-Dickinson) containing 15 ml of culture medium [medium 199 with Earle’s salts, 2.3 g/l NaHCO3, amino acids, and Glutamax (Life Technologies), supplemented with antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) (Life Technologies), and 10% (v/v) heat-inactivated fetal calf serum (Boehringer Ingelheim)]. At confluence, HUVECs were treated with a solution of 0.05% trypsin-0.02% EDTA (Life Technologies). At the first passage, they were cultured in the above growth medium plus 100 µg/ml endothelial cell growth supplement (Sigma) and 90 µg/ml heparin (Sigma). HUVECs and ECV304 cells were grown at 37°C in a humidified atmosphere containing 5% CO2.

Cell treatments
Hcy, dexamethasone, acivicin, and antitumoral drugs (daunorubicin, VP-16, vinblastine, Ara-C) were purchased from Sigma (L’Isle d’Abeau-Chesnes, France) and Cu2+ (in the form of copper sulfate) was provided by Prolabo (Fontenay sous Bois, France). Stock solutions of Hcy, dexamethasone, acivicin, and Cu2+ were prepared in culture medium at 10 mmol/l, 1 mmol/l, 100 µmol/l, and 4 mmol/l, respectively. Stock solutions of antitumoral drugs were prepared as follows: daunorubicin, VP-16, vinblastine, and Ara-C were dissolved in dimethyl sulfoxide (Sigma) at 10 mmol/l, 20 mmol/l, 10 mg/ml, and 50 mmol/l, respectively. Hcy was used at 0.1, 0.5, and 1 mmol/l final concentrations and Cu2+ at 4, 10, and 20 µmol/l final concentrations. Hcy was added first in the culture medium of ECV304 cells or HUVECs precultured for various periods of time; Cu2+ was added immediately after. Treatments with Hcy, Cu2+, and Hcy plus Cu2+ were performed for 24 h. Dexamethasone was used at 5 nmol/l final concentration. Acivicin was used at 2 µmol/l and 1.5 µmol/l final concentrations in ECV304 cells and HUVECs, respectively. Dexamethasone and acivicin were introduced into the culture medium 1 h before Hcy (1 mmol/l) plus Cu2+ (10 µmol/l) or antitumoral drugs (daunorubicin: 2 µmol/l; VP-16: 50 µmol/l; vinblastine: 20 nmol/l; Ara-C: 100 µmol/l). Z-VAD-fmk; Bachem Biochimie, Voisins-le-Bretonneux, France), a broad-spectrum, cell-permeable caspase inhibitor (45) , was dissolved in dimethyl sulfoxide (Sigma) at 2 mmol/l, and added to the culture medium at 100 µmol/l final concentration 30 min before Hcy (1 mmol/l) plus Cu2+ (10 µmol/l).

Cell counting
Cell counting of adherent and nonadherent cells was performed with an hematocytometer under an inverted Laborlux IX70 phase contrast microscope (Olympus, Tokyo, Japan) on cells cultured in 6-well plates (Falcon/Becton-Dickinson, Plymouth, UK). Adherent cells were collected by trypsinization with a solution of 0.05% trypsin-0.02% EDTA (Life Technologies). Cell detachment, which constitutes an index of cytotoxicity, was quantified (51) .

Determination of cell permeability with PI
Cell permeability was determined after staining with PI ({lambda}Ex max: 540 nm, {lambda}Em max: 625 nm), which stains only dead cells (44 , 52) . PI was used at 5 µg/ml on the cell suspension adjusted to 106 cells/ml and the fluorescence was immediately quantified by flow cytometry with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The red fluorescence of PI was collected through a 585/42 nm band-pass filter and the fluorescence signals were measured on a logarithmic scale. For each sample, 10,000 cells were acquired and the data were analyzed with LYSYS I software (Becton Dickinson).

Identification and quantification of apoptotic cells after nuclei staining with Hoechst 33342
Nuclear morphology of control and treated cells was characterized by fluorescence microscopy after staining with Hoechst 33342 (Sigma); apoptotic cells were characterized by nuclear condensation of chromatin and/or nuclear fragmentation (44) . Hoechst 33342 was added to the culture medium at a final concentration of 10 µg/ml; after 30 min of incubation at 37°C, adherent cells were collected by trypsinization with a solution of 0.05% trypsin-0.02% EDTA (Life Technologies), mixed with nonadherent cells, and resuspended at a concentration of 106 cells/ml in cold PBS containing 2% (w/v) paraformaldehyde. After 15 min, cell deposits of ~40,000 cells were applied to glass slides by cytocentrifugation (5 min, 15,000 rpm) with a cytospin 2 (Shandon, Cheshire, UK), mounted in Fluoprep (Biomérieux, Marcy l’Etoile, France), coverslipped, and stored in the dark at 4°C. The morphological aspect of cell nuclei was observed with an Axioskop right microscope (Zeiss, Jena, Germany) by using UV light excitation. Images were acquired with an image analysis system (Biocom, Les Ulis, France) and 300 cells were examined for each sample.

Flow cytometric measurement of mitochondrial transmembrane potential ({Delta}{Psi}m) with the dye DiOC6(3)
Variations of the mitochondrial transmembrane potential ({Delta}{Psi}m) during Hcy plus Cu2+-induced apoptosis were studied with 3, 3'-dihexyloxacarbocyanine iodide (DiOC6(3) ({lambda}Ex max 484 nm, {lambda}Em max 501 nm) (Molecular Probes, Eugene, OR). DiOC6(3), which accumulates in the mitochondrial matrix under the influence of the {Delta}{Psi}m (53) , was used at a final concentration of 40 nmol/l on cell suspensions adjusted to 2 x 106 cells/ml. After 15 min of incubation at 37°C, the DiOC6 (3) transmembrane-mitochondrial potential-related fluorescence was immediately recorded by flow cytometry with a GALAXY flow cytometer (Dako, Trappes, France). The green fluorescence was collected through a 520/10 nm band-pass filter and the fluorescent signals were measured on a logarithmic scale. For each sample, 10,000 cells were acquired and the data were analyzed with the FlowMate software (Dako).

Western blot analysis of mitochondrial cytochrome c release
Cytochrome c release from the intermembrane space of mitochondria into the cytosol was investigated by Western blot analysis in ECV304 cells incubated for 24 h in culture medium in the presence of Hcy (1 mmol/l) plus Cu2+ (10 µmol/l). At the end of the treatment, nonadherent and adherent cells were harvested, washed twice with ice-cold PBS, and resuspended in buffer A (20 mmol/l HEPES-KOH, pH 7.5, 10 mmol/l KCl, 1.5 mmol/l MgCl2, 1 mmol/l sodium EDTA, 1 mmol/l sodium EGTA, 1 mmol/l dithiothreitol) containing 250 mmol/l sucrose and a mixture of protease inhibitors (1 mmol/l PMSF, 1% aprotinin, 1 mmol/l leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml chymostatin). The cells were further homogenized. Unbroken cells, large plasma membrane pieces, and nuclei were removed by centrifuging the homogenates at 1000 g at 4°C for 10 min. The resulting supernatant was subjected to 10,000 g centrifugation at 4°C for 20 min. The supernatant was recentrifuged at 100,000 g (4°C, 1 h) to generate cytosol.

The protein concentration was measured by using bicinchoninic acid reagent (Pierce, Rockford, IL) (54) . Fifty microgram proteins were incubated in loading buffer (125 mmol/l Tris-HCl, pH 6.8, 10% ß-mercapto-ethanol, 4.6% SDS, 20% glycerol and 0.003% bromphenol blue), separated by SDS-PAGE, and electroblotted to PVDF membrane (Bio-Rad, Ivry sur Seine, France). After blocking nonspecific binding sites overnight by 5% nonfat milk in TPBS (PBS, 0.1% Tween 20), the PVDF membrane was successively incubated for 2 h at room temperature with a mouse monoclonal antibody directed against cytochrome c (PharMingen, San Diego, CA), washed twice in TPBS, incubated with an horseradish peroxidase-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at room temperature, and washed twice more in TPBS. The immunoblot was revealed by autoradiography using an enhanced chemiluminescence detection kit (Amersham, Les Ulis, France). The autoradiograph was photographed and computerized with an image analysis system (Biocom, Les Ulis, France). Each experiment was repeated three or four times with identical results.

Identification of the active form of caspase-3 and of poly(ADP-ribose) polymerase degradation by immunocytochemistry
Detection of active caspase-3 and of cleaved poly(ADP-ribose)polymerase (PARP) was performed on cell deposits of ~40,000 ECV304 cells applied to silanated glass slides by cytocentrifugation for 5 min at 15,000 rpm with a cytospin 2 (Shandon) and stored at -20°C. For immunocytochemistry, after blocking of the endogenous peroxidase activity with 0.33% H2O2 for 15 min at room temperature, the slides were washed in phosphate-buffered saline adjusted to pH 7.2 (PBS) and incubated for 15 min at room temperature with normal goat serum (BIOSPA, Milano, Italy) diluted 1:30 in PBS. After three washes in PBS, the slides were incubated for either 1 h at room temperature with the polyclonal rabbit anti-active caspase-3 antibody (PharMingen), diluted 1 µg/ml in PBS, or overnight at 4°C with the polyclonal rabbit antibody raised against cleaved PARP (New England Biolabs, Beverly, MA) diluted 1:100 in PBS. After three washes in PBS, the slides were successively incubated for 15 min at room temperature with a biotinylated goat anti-rabbit IgG (BIOSPA), diluted 1:250 in PBS, washed three times in PBS, and incubated for 15 min with streptavidin peroxidase complex (BIOSPA), diluted 1:250 in PBS. After washing in PBS, peroxidase activity was revealed with 3, 3'-diaminobenzidine (Dako, Copenhagen, Denmark) for 5 to 10 min at room temperature. Counterstaining was performed with eosin and methylene blue (RAL 555 kit, CML, Nemours, France); slides were mounted in Eukitt (CML) and stored at room temperature until examination with an Axioskop right microscope (Zeiss). The controls used included the omission of the primary antibodies (conjugated controls); under this condition, no aspecific signals were observed. Cells were examined under an Axioskop microscope (Zeiss) and images were computerized with an image analysis system (Biocom).

DNA fragmentation assay on agarose gel
DNA fragmentation assays were performed by electrophoresis on 1.8% agarose gel. Cellular DNA was extracted as described previously (40) by using a DNA extraction kit (Stratagene, La Jolla, CA). After electrophoresis, gels were examined under ultraviolet light and computerized with an image analysis system (Biocom).

Quantification of reduced cellular glutathione
Quantification of reduced glutathione (GSH) was performed on cell lysates with Calbiochem Glutathione Assay Kit (CALBIOCHEM, San Diego, CA) according to the manufacturer’s procedure. Cell lysates were obtained from 3 x 106 cells suspended in 500 µl of a 5% (w/v) metaphosphoric acid solution (Sigma) and homogenized by successive pipetting. After centrifugation at 3000 g (10 min,+4°C), 300 µl of the resulting supernatant (cell lysate) were collected and mixed with 600 µl of a buffer solution (200 mmol/l potassium phosphate (pH 7.8) containing 0.2 mmol/l diethylene triamine pentaacetic acid and 0.025% (v/v) LUBROL), 50 µl of chromogenic reagent (12 mmol/l of 4-chloro-1-methyl-7-trifluoromethyl quinolone methylsulfate in 0.2 N HCl), and 50 µl of a 30% (w/v) NaOH solution. After 10 min of incubation in the dark at 25°C, the absorbance of the samples was immediately read at 400 nm with a DUR-64 spectrophotometer (Beckman Instruments, Fullerton, CA); the quantity of GSH (µmol/106 cells) was determined comparatively to a standard curve performed with GSH (Sigma) in the same experimental conditions.

Quantification of cellular {gamma}-GT activity
The enzyme {gamma}-GT (EC 2.3.2.2), associated with the outer leaflet of the plasma membrane, catalyzes the first step in the extracellular transpeptidation of the main cellular antioxidant glutathione via the {gamma}-glutamyl cycle into amino acid intermediates (39 , 41) . These amino acid intermediates are subsequently transported across the cell membrane and used in the de novo synthesis of intracellular glutathione. Quantification of the {gamma}-GT activity was performed on whole cells with the use of a Sigma Diagnostics colorimetric assay (Sigma). To this end, 50 µl of cell suspension (corresponding to 106 cells in 50 µl of PBS) was mixed with 500 µl of substrate solution [51 µmol/l {gamma}-glutamyl-p nitroanilide and 1.1 mmol/l glycylglycine dissolved in Tris buffer (0.1 mol/l, pH 7.2)] and incubated at 37°C for 20 min. Thereafter, 2 ml of acetic acid solution (50 ml of glacial acetic acid dissolved in 450 ml of distilled water) and 1 ml of sodium nitrite solution (7.5 mmol/l sodium nitrite in distilled water) were added (for the blank, the cell suspension was introduced after the addition of the acetic acid solution). After 3 min at room temperature, 1 ml of a 0.1% (w/v) ammonium sulfamate solution was added; after a subsequent incubation of 3 min at room temperature, each sample was mixed with 1 ml of N-1-naphtylethylenediamine solution (2 mmol/l N-1-naphtylethylenediamine in distilled water). The absorbance of the sample was immediately read at 550 nm with a DUR-64 spectrophotometer (Beckman Instruments) and the enzymatic activity (nmol/min/106 cells) was determined in comparison to a standard curve performed in the same experimental conditions with a {gamma}-glutamyl transpeptidase calibration solution (Sigma).

Statistical methods
Statistical analyses were performed with SYSTAT software (Evanston, IL) by using either a one-way ANOVA, followed by a Dunnett t test, or a two-way ANOVA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Influence of homocysteine and copper on cell growth and cell permeability to PI
The effects of Hcy plus Cu2+ on cell growth and cell permeability to PI (which enters in dead cells only; ref52 ) were investigated in ECV304 cells cultured for 3 days, then treated with Hcy plus Cu2+ for an additional 24 h. Hcy concentrations chosen (0.1, 0.5, and 1 mmol/l) were in the range of those found in the plasma of patients with severe hyperhomocysteinemia and corresponded to free plus albumin-bound Hcy (36 , 55 , 56) . However, in our experimental conditions, albumin concentration was lower (3 g/l) in the culture medium than in human plasma (40 g/l). Therefore, the proportion of free Hcy was probably higher in our experiments than in physiological conditions, whereas formation of homocystine and of homocysteine-cysteine mixed disulfide cannot be excluded and could also contribute to reduced free Hcy (5) . Cu2+ concentrations (4, 10, and 20 µmol/l) were also in the range of those usually found in human plasma (57) . Cu2+ was added to the culture medium in the form of copper sulfate since no difference of cytotoxicity had been reported when endothelial cells were exposed to Hcy plus Cu2+ used in the form of copper sulfate or of ceruloplasmin, which is the physiological carrier of Cu2+ (35) .

Hcy and Cu2+ taken separately had no effect on cell growth characteristics whatever the concentrations under these conditions. Indeed, compared with untreated cells, Hcy and Cu2+ did not significantly modify the number of adherent and nonadherent cells (Fig. 1A ) or the proportions of PI-permeable cells (Fig. 1B ). However, when Hcy plus Cu2+ were combined, cell growth characteristics were strongly modified (Fig. 1A , B ): cell growth was reduced, cellular detachment was favored, and cell permeability to PI was enhanced. Therefore, when ECV304 cells were treated with Hcy plus Cu2+, a concentration-dependent decrease of cell proliferation was observed and the total number of cells (adherent plus nonadherent cells) in Hcy plus Cu2+ wells was much lower than the total number of cells in controls in the absence of either Hcy, Cu2+, or both (Fig. 1A ). After treatment with Hcy plus Cu2+, loss of cell adhesion was increased as shown by significantly (P<0.05) higher numbers of nonadherent cells floating in the culture medium (Fig. 1A ) and a gradual enhancement of cells permeable to PI was observed (Fig. 2A ).



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  		Figure 1. Effects of homocysteine, copper, and homocysteine plus copper on cell growth and permeability to propidium iodide. The effects of homocysteine (Hcy: 0.1, 0.5, and 1 mmol/l) plus copper (Cu2+) (4, 10, and 20 µmol/l) on cell growth and cell permeability to propidium iodide (PI) were investigated in ECV304 cells precultured for 3 days and subsequently treated for an additional 24 h with either Hcy, Cu2+, or Hcy plus Cu2+. ECV304 cells were seeded at a concentration of 0.18 x 105 cells in 3 ml of culture medium per well of 6-well plates. The number of adherent and nonadherent cells (per well of 6-well plates) was determined with the use of hematocytometer. The percentage of cells permeable to PI was quantified by flow cytometry. Untreated and treated cells are represented by different symbols. Open and filled symbols correspond to adherent and nonadherent cells, respectively; square: untreated cells; circle: Hcy (0.1, 0.5, 1 mmol/l); triangle: Cu2+ (4, 10, and 20 mmol/l); inverted triangle: Hcy (0.1, 0.5, 1 mmol/l) plus Cu2+ (4 µmol/l); multiplied symbol: Hcy (0.1, 0.5, 1 mmol/l) plus Cu2+ (10 µmol/l); cross: Hcy (0.1, 0.5, 1 mmol/l) plus Cu2+ (20 µmol/l). Data are mean ± SD of 3 independent experiments performed in triplicate. Significance of the difference between untreated and treated cells (*P<0.05).



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  		Figure 2. Microscopical characterization and quantification of cell death after treatment with homocysteine plus copper. ECV304 cells precultured for 3 days were subsequently treated for an additional 24 h with either Hcy (0.1, 0.5, and 1 mmol/l), copper (Cu2+) (4, 10, and 20 µmol/l), or Hcy plus Cu2+. Cells were observed by fluorescence microscopy to characterize the morphological aspects of cell nuclei after staining with Hoechst 33342. Fluorescence microscopy (x700) of untreated (A) and Hcy (1 mmol/l) plus Cu2+ (10 µmol/l) -treated cells (B). In Hcy plus Cu2+-treated cells, some cells with fragmented and/or condensed nuclei characteristic of apoptotic cells were observed; in untreated cells and Hcy- or Cu2+-treated cells (data not shown), most had round and regular nuclei. C) Percentages of apoptotic cells (defined on adherent plus nonadherent cells) were determined by fluorescence microscopy after nuclei staining with Hoechst 33342 on cell deposits performed by cytocentrifugation on glass slides. Untreated and treated cells are represented by different symbols; square: untreated cells; circle: Hcy (0.1, 0.5, 1 mmol/l); triangle: Cu2+ (4, 10, and 20 mmol/l); inverted triangle: Hcy (0.1, 0.5, 1 mmol/l) plus Cu2+ (4 µmol/l); multiplied symbol: Hcy (0.1, 0.5, 1 mmol/l) plus Cu2+ (10 µmol/l); cross: Hcy (0.1, 0.5, 1 mmol/l) plus Cu2+ (20 µmol/l). Data are mean ± SD of 3 independent experiments performed in triplicate. Significance of the difference between untreated and treated cells (*P<0.05).

Microscopic characterization and quantification of nuclear changes occurring after treatment with homocysteine plus copper
ECV304 cells precultured for 3 days were treated for an additional 24 h in the presence of either Hcy (0.1, 0.5, and 1 mmol/l), Cu2+ (4, 10, and 20 µmol/l), or Hcy plus Cu2+. Adherent and nonadherent cells were then collected and stained with Hoechst 33342, which identifies apoptotic cells characterized by condensed and/or fragmented nuclei (44) . The proportions of apoptotic cells present in untreated cells and in Hcy or Cu2+-treated cells were low and similar, accounting for 1 ± 1% to 2 ± 1% of total cells (Fig. 2A ). However, compared with untreated cells, the proportions of apoptotic cells in the presence of Hcy plus Cu2+ were always significantly (P<0.05) enhanced (Fig. 2B ). In these later conditions, at the lowest Hcy concentration studied (0.1 mmol), the proportions of apoptotic cells were slightly increased (3±1% to 6±2%) whereas at highest Hcy concentrations (0.5 and 1 mmol/l), the proportions of apoptotic cells were strongly enhanced and varied from 15 ± 2% to 48 ± 2% (Fig. 2C ).

For further characterization of apoptosis, Hcy was added to the culture at 1 mmol/l. Indeed, this concentration gave the highest proportions of apoptotically dying cells when combined with Cu2+ used at concentrations of 4, 10, and 20 µmol/l, yielding a sufficient number apoptotic cells to investigate their characteristics. As for Cu2+, it was used at 10 µmol/l since this concentration is commonly found in human plasma (57) .

Characterization of homocysteine plus copper-induced apoptosis
Apoptosis is a complex phenomenon that can involve different metabolic pathways depending on the inducer of cell death considered. In the case of chemical-induced apoptosis, the cascade of events leading to cell death generally induces a loss of transmembrane mitochondrial potential ({Delta}{Psi}m) accompanied by a translocation into the cytosol of some mitochondrial proteins such as cytochrome c, apoptosis-inducing factor, and SMAC/DIABLO (58) . Among these proteins, cytochrome c contributes in activating a cascade of caspases, such as caspase-3, which participates to the degradation of numerous nuclear proteins including PARP, leading subsequently to the internucleosomal cleavage of DNA (58 , 59) . In the present work, ECV304 cells were precultured for 3 days and subsequently treated for an additional 24 h in their culture medium alone or in the presence of Hcy (1 mmol/l), Cu2+ (10 µmol/l), or Hcy plus Cu2+. Under these conditions, the loss of {Delta}{Psi}m and the processing of cytochrome c release were simultaneously evaluated in ECV304 cells (including adherent and nonadherent cells) by flow cytometry with the cationic lipophilic dye DiOC6(3) and by Western blot with a mouse monoclonal antibody directed against cytochrome c, respectively. When compared with untreated cells, treatment with Hcy plus Cu2+ resulted in a loss of {Delta}{Psi}m (Fig. 3 ) and a cytosolic release of cytochrome c (the rising level of cytochrome c release into the cytosol was estimated in comparison with the constant level of a cross-reacting protein of ~65 kDa present in the cytosolic extract) (Fig. 3) . As mitochondrion generally transduces some pro apoptotic stimuli that trigger activation of a cascade of caspases (60 , 61) , the role of these enzymes in Hcy plus Cu2+-induced cell death was evaluated with the use of Z-VAD-fmk (100 µmol/l), which is a wide-spectrum caspase inhibitor (45) . The involvement of caspases in Hcy plus Cu2+-induced cell death was supported by the ability of Z-VAD-fmk to restore a proportion of apoptotic cells similar to those found in untreated cells (Table 1 ). Z-VAD-fmk was also capable of totally inhibiting cell detachment induced by Hcy plus Cu2+, whereas it only partially decreased the proportion of cells permeable to PI (Table 1) . Among the different caspases that can play a role in Hcy plus Cu2+-induced apoptosis, the involvement of caspase-3 was shown by immunocytochemistry with the use of a polyclonal rabbit antibody raised against active caspase-3 consisting of 17 and 12 kDa subunits derived from the 32 kDa inactive proenzyme (62) . Immunocytochemistry was also chosen to investigate PARP degradation depending on the caspase-3 activity (62) . A polyclonal rabbit antibody detecting only cleaved PARP (89 kDa carboxyl-terminal catalytic domain) was used. Under these conditions, active caspase-3 and PARP degradation was observed only in cells with fragmented and/or condensed nuclei (Fig. 3) . After treatment with Hcy plus Cu2+, the mode of DNA degradation was assessed by electrophoresis on a 1.8% agarose gel. A typical internucleosomal DNA fragmentation in 180 to 200 bp and multiples characteristic of cell death by apoptosis were observed (Fig. 3) . In untreated cells and in the presence of Hcy or of Cu2+ alone, no apoptotic cells were identified (Table 1) and consequently no loss of {Delta}{Psi}m, no cytosolic release of cytochrome c, no active form of caspase-3, no PARP degradation, and no internucleosomal DNA fragmentation were observed (Fig. 3) .



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  		Figure 3. Characteristics of homocysteine plus copper-induced apoptosis. ECV304 cells precultured for 3 days were treated for an additional 24 h either in their culture medium alone or in the presence of Hcy (1 mmol/l), copper (Cu2+) (10 µmol/l), or Hcy plus Cu2+. Under these conditions, the transmembrane mitochondrial potential ({Delta}{Psi}m) was measured with DiOC6(3) (A), cytosolic release of cytochrome c was determined by Western blot (B), the presence of active caspase-3 and of PARP degradation was analyzed by immunocytochemistry (C–E), and the DNA fragmentation pattern was investigated on agarose gel (F). A) Fluorescence associated with DiOC6(3) was measured by flow cytometry and 10,000 cells (adherent plus nonadherent cells) were analyzed for each assay; green, red, and black lines correspond to the fluorescence associated with DiOC6(3) in untreated cells, Hcy-treated cells, and Cu2+-treated cells, respectively; the red histogram corresponds to fluorescence associated with DiOC6(3) in Hcy plus Cu2+-treated cells. Similar values of {Delta}{Psi}m (similar fluorescence intensities) were found in untreated-, Hcy-, and Cu2+-treated cells whereas a loss of {Delta}{Psi}m was detected in Hcy plus Cu2+-treated cells. B) The rising level of the release of cytochrome c into the cytosol from adherent plus nonadherent cells was estimated vs. the constant level of a cross-reacting protein of ~65 kDa present in the cytosolic extract. A cytosolic release of cytochrome c was only observed in Hcy plus Cu2+-treated cells; lane 1: untreated cells; lane 2: Hcy-treated cells; lane 3: Cu2+-treated cells; lane 4: Hcy plus Cu2+-treated cells. Cells containing active caspase-3 (D) and cleaved PARP (E) are stained in brown; such cells were observed only after treatment with Hcy plus Cu2+ and not in untreated cells (C) or in Hcy- or Cu2+-treated cells (data not shown) (x700). Internucleosomal DNA fragmentation (F) was observed after treatment with Hcy plus Cu2+ (lane 4), whereas no DNA ladder was detected in untreated (lane 1), Hcy- (lane 2), or Cu2+- (lane 3) treated cells. Data shown are representative of 3 independent experiments.


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  		Table 1. Inhibition of homocysteine plus copper-induced apoptosis by Z-VAD-fmka

Influence of the period of culture before treatment on the ability of homocysteine plus copper to induce cell death
To investigate the influence of the length of the culture period before treatment on the ability of Hcy plus Cu2+ to induce cell death, ECV304 cells were cultured for 3, 4, or 5 days in their culture medium alone. ECV304 cells were incubated for an additional 24 h in either their culture medium alone (untreated cells) or in the presence of Hcy (1 mmol/l), Cu2+ (10 µmol/l), or Hcy plus Cu2+ Similar and low percentages (below 5%) of cell detachment, PI-positive cells, and apoptotic cells (characterized by fragmented and/or condensed nuclei) were identified in untreated cells and after treatment with Hcy or Cu2+ (Fig. 4 ). In the presence of Hcy plus Cu2+, the percentages of cell detachment, PI-positive cells, and apoptotic cells were highest when the cells had been cultured for only 3 days before treatment (Fig. 4) . Therefore, the cytotoxicity of Hcy plus Cu2+ depends on the length of time of culture before treatment.



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  		Figure 4. Influence of the time of culture on the cytotoxicity of homocysteine plus copper. ECV 304 cells precultured for 3, 4, or 5 days were subsequently treated for an additional 24 h either in their culture medium alone or in the presence of homocysteine (1 mmol/l), copper (Cu2+) (10 µmol/l), or Hcy plus Cu2+. The percentage of cell detachment [number of nonadherent cells/(number of adherent plus nonadherent cells)] was determined with the use of hematocytometer, the percentage of cells permeable to propidium iodide was quantified by flow cytometry, and the percentage of apoptotic cells characterized by fragmented and/or condensed nuclei after staining with Hoechst 33342 was defined by fluorescence microscopy. Data are mean ± SD of 3 independent experiments performed in triplicate. *Significance of the difference between untreated and treated cells (*P < 0.05).

Influence of the time of culture on {gamma}-GT activity
The cytotoxicity of Hcy was described to depend on the redox status of the cells and to decrease the level of intracellular glutathione (21 , 36) . Since the ability of Hcy plus Cu2+ depends on the length of time of culture before treatment, we asked whether {gamma}-GT activity, which is implied in glutathione metabolism (39 , 41) and has been associated with resistance to various antitumoral drugs (42 , 43) , varied with the time of culture. ECV304 cells were cultured for 3, 4, and 5 days in their culture medium alone. At these different times of culture, {gamma}-GT activity and the level of GSH per 106 cells were determined (Fig. 5 ). {gamma}-GT activity and the level of GSH increased with the time of culture. Therefore, the highest {gamma}-GT activity and the highest level of GSH were measured at 5 days of culture.



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  		Figure 5. Influence of the time of culture on {gamma}-glutamyl transpeptidase activity and glutathione content. ECV 304 cells were cultured for 3, 4, and 5 days in their culture medium alone. At these different culture times, {gamma}-GT activity and the level of reduced glutathione (GSH) were determined. Data are mean ± SD of 3 independent experiments performed in triplicate.

Difference of toxicity of homocysteine plus copper according to {gamma}-GT activity
When the period of culture before treatment increases, the ability of Hcy (1 mmol/l) plus Cu2+ (10 µmol/l) to induce apoptosis decreases (Fig. 4) , whereas {gamma}-GT activity increases (Fig. 5) . Therefore, we attempted to demonstrate that {gamma}-GT activity was implied in the control of cell death when ECV304 cells and HUVECs were treated with Hcy plus Cu2+. The influence of {gamma}-GT activity was investigated with the use of dexamethasone (5 nmol/l) and acivicin (used at 2 µmol/l in ECV304 cells and at 1.5 µmol/l in HUVECs), which stimulates and inhibits {gamma}-GT activity, respectively. The ability of dexamethasone to stimulate {gamma}-GT activity was investigated in cells taken at an early time of culture as they have moderate {gamma}-GT activity whereas the ability of acivicin to counteract {gamma}-GT activity was studied in cells cultured for a longer period, as they have a higher level of {gamma}-GT activity (Fig. 5 , Table 2 ).


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  		Table 2. Influence of {gamma}-GT activity on the toxicity of homocysteine plus copper on HUVECsa

To stimulate {gamma}-GT activity, ECV304 cells and HUVECs (taken at the first passage) were cultured for 3 days in their culture medium alone and for an additional 24 h in the absence or presence of dexamethasone. Compared with untreated cells maintained for 4 days in their culture medium alone, {gamma}-GT activity and GSH content per cell increased significantly under these conditions from treatment with dexamethasone (Fig. 6A -B , Table 2 ). Hcy plus Cu2+ decreased {gamma}-GT activity as well as GSH content per cell, and these effects were counteracted by dexamethasone (Fig. 6A , B , Table 2 ). When ECV304 cells and HUVECs were previously maintained for 3 days in their culture medium alone and incubated for an additional 24 h with dexamethasone and Hcy plus Cu2+, they were less sensitive to the occurrence of cell death by apoptosis (characterized by the presence of cells with fragmented and/or condensed nuclei) than cells cultured in the presence of Hcy plus Cu2+ without dexamethasone (Fig. 6C , Table 2 ). Dexamethasone was also capable of reducing the loss of cell adhesion and increased permeability to PI in HUVECs (Table 2) . So, stimulation of {gamma}-GT activity with dexamethasone (associated with an increase of GSH) preserves ECV304 cells and HUVECs from Hcy plus Cu2+-induced apoptosis.



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  		Figure 6. Difference of toxicity of homocysteine plus copper according to {gamma}-GT activity modulated with dexamethasone and acivicin. The influence of {gamma}-GT activity was investigated with the use of dexamethasone (5 nmol/l) and acivicin (2 µmol/l), which stimulates and inhibits {gamma}-GT activity, respectively. To stimulate or inhibit {gamma}-GT activity, cells were cultured 3 or 5 days in their culture medium alone and for an additional 24 h in the absence or presence of dexamethasone or acivicin, alone or in combination with homocysteine (Hcy) (1 mmol/l) plus copper (Cu2+) (10 µmol/l). Effects of dexamethasone and acivicin are shown on {gamma}-GT activity (A) and GSH content (B). C) The ability of dexamethasone and acivicin to inhibit or stimulate apoptosis, respectively. Data are mean ± SD of 3 independent experiments performed in triplicate. Significance of the differences between untreated and dexamethasone- or acivicin-treated cells (*P<0.05); significance of the differences between untreated and Hcy plus Cu2+-treated cells (**P<0.05); significance of the differences between Hcy plus Cu2+-treated cells and Hcy plus Cu2+ plus dexamethasone or acivicin-treated cells (***P<0.05).

To inhibit {gamma}-GT activity, ECV304 cells and HUVECs were cultured for 5 days in their culture medium alone and for an additional 24 h in the absence or presence of acivicin. Compared with untreated cells maintained for 6 days in their culture medium alone, {gamma}-GT activity and GSH content per 106 cells significantly decreased with acivicin (Fig. 6A , B , Table 2 ). Hcy plus Cu2+ contributes to decrease {gamma}-GT activity, as well as GSH content per cell, and these effects were potentialized by acivicin (Fig. 6A , B ). In HUVECs, acivicin also favored the loss of cell adhesion and increased permeability to PI (Table 2) . When ECV304 cells and HUVECs were previously maintained for 5 days in their culture medium alone and incubated for an additional 24 h with acivicin and Hcy plus Cu2+, they were more sensitive to apoptosis than cells cultured with Hcy plus Cu2+ without acivicin (Fig. 6C ). Therefore, inhibition of {gamma}-GT activity with acivicin (associated with a decrease of GSH) favors apoptosis induced by Hcy plus Cu2+.

Taken together, these data demonstrate that the level of {gamma}-GT activity modulates Hcy plus Cu2+-induced apoptosis on both ECV304 cells and HUVECs.

Differences of toxicity of various antitumoral drugs (daunorubicin, VP-16, vinblastine, and Ara-c) according to {gamma}-GT activity
Experimental conditions similar to those used with Hcy plus Cu2+ in the absence or presence of dexamethasone (5 nmol/l) or acivicin (2 µmol/l) were chosen to investigate (in ECV304 cells) the influence of {gamma}-GT activity on the cytotoxicity of various antitumoral drugs with unrelated structures and mechanisms of action ]daunorubicin is an anthracycline with antineoplasic and antibiotic properties that intercalates in adjacent bases of the DNA leading to inhibition of DNA replication (63) , VP-16 belongs to the class of epipodophyllotoxin and is an inhibitor of topoisomerase II (64) , vinblastine is an alkaloid that blocks the formation of microtubules involved in the formation of the mitotic spindle (65) , and Ara-C is an antimetabolite that is a structural analog to pyrimidic bases and incorporates into the DNA leading to inhibition of DNA synthesis (66) ].

Thus, when the cells were previously maintained for 3 days in their culture medium alone and incubated for an additional 24 h with dexamethasone and antitumoral drugs (daunorubicin: 2 µmol/l; VP-16: 50 µmol/l; vinblastine: 20 nmol/l; Ara-C: 100 µmol/l), they were less sensitive to apoptosis (characterized by the occurrence of cells with fragmented and/or condensed nuclei) than their counterparts cultured with antitumoral drugs without dexamethasone (Fig. 7A ). On the contrary, when the cells were treated for 5 days in culture medium alone and then incubated for an additional 24 h with acivicin and antitumoral drugs, they were more sensitive to apoptosis than the cells cultured in the presence of antitumoral drugs without acivicin (Fig. 7B ). Thus, stimulation of {gamma}-GT activity with dexamethasone inhibits daunorubicin-, VP-16-, vinblastine-, and Ara-C-induced apoptosis whereas inhibition of {gamma}-GT activity with acivicin favors cell death induced by these different antitumoral drugs. Therefore, these data lead us to suppose that {gamma}-GT activity could play an important role in the control of apoptosis whatever the proapoptotic chemical agent considered.



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  		Figure 7. Difference of toxicity of antitumoral drugs [daunorubicin, etoposide (VP-16), vinblastine, and cytosine ß-D-arabinofuranoside (Ara-C)] according to {gamma}-GT activity modulated with dexamethasone and acivicin. The influence of {gamma}-GT activity was investigated in ECV304 cells treated with different antitumoral drugs (daunorubicin: 2 µmol/l; VP-16: 50 µmol/l; vinblastine: 20 nmol/l; Ara-C: 100 µmol/l). Cells were cultured 3 or 5 days in their culture medium alone and for an additional 24 h in the absence or presence of dexamethasone (used at 5 nmol/l to stimulate {gamma}-GT activity) (A) or acivicin (used at 2 µmol/l to inhibit {gamma}-GT activity) (B) either alone or in combination with antitumoral drugs. Data are mean ± SD of 3 independent experiments performed in triplicate. Significance of the differences between untreated and treated cells (*P<0.05); significance of the differences between antitumoral drug-treated cells and (antitumoral drugs plus dexamethasone or acivicin) -treated cells (**P<0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Atherosclerosis is a complex degenerative pathology that can involve physical, chemical, and/or biological agents (2) . During the past few years, there has been accumulating evidence that Hcy (an intermediate compound formed during the metabolism of methionine) (5) constitutes an independent risk factor associated with atherosclerotic vascular disease (67) . Indeed, patients with high plasma levels of Hcy (higher than 0.1 mmol/l) rapidly develop atherosclerosis and have an increased risk of vascular occlusion (3 , 4) . Thus, it is important to understand the mechanism of vascular injury associated with this agent whose cytotoxicity could be due, at least in part, to inhibition of methylation (27) , protein homocysteinylation (68) , and altered gene expression (69) . Since apoptosis may be an important process in atheromatous lesions in both human (70) and animal models (71 72 73) , we asked whether Hcy was able to induce this mode of cell death.

In the present study, we report that Hcy plus Cu2+ was able to induce an apoptotic process in ECV304 cells and on HUVECs. This form of cell death by apoptosis was characterized by an inhibition of cell growth, a loss of cell adhesion, an increased permeability to PI, a drop of transmembrane mitochondrial potential ({Delta}{Psi}m), a cytosolic release of cytochrome c, an activation of caspase-3, a degradation of PARP, an internucleosomal DNA fragmentation, and the occurrence of cells with fragmented and/or condensed nuclei whereas Hcy and Cu2+ taken separately had no cytotoxic effects. We linked the apoptotic potency of Hcy plus Cu2+ with {gamma}-GT activity and demonstrated that the ability of Hcy plus Cu2+ to induce apoptosis was inversely proportional to the activity of this enzyme.

Quantification of the number of adherent and nonadherent cells and a determination of the proportions of cells permeable to PI are frequently used to investigate the toxic effects on adherent cells, (44 , 51) . These criteria were chosen to define whether cellular injury could occur after treatment with Hcy. In agreement with previous data performed on human and bovine endothelial cells as well as on human histiocytic cells U937, by using similar Hcy concentrations (35 , 74 , 75) , the results of this study performed in ECV304 cells and HUVECs underscore the absence of toxicity of Hcy taken individually (75 , 76) and confirm the ability of Hcy plus Cu2+ to trigger cell death characterized by a loss of cell adhesion and increased permeability to PI. Since different forms of cell death are described (30) , the mode of cell death triggered by Hcy plus Cu2+ was characterized and defined as an apoptotic process involving caspases. Indeed, after treatment with Hcy plus Cu2+, an increased proportion of cells with morphological characteristics of apoptosis (cells with fragmented and/or condensed nuclei) (44) was observed. In addition, Z-VAD-fmk (a wide-spectrum caspase inhibitor) (45) was able to counteract Hcy plus Cu2+-induced cytotoxicity (mainly cell detachment and morphological changes of cell nuclei, and partially enhanced permeability to PI). Because caspases (a family of cysteine proteases with a substrate specificity for Asp residue that are integral part of numerous apoptotic processes) (61) can be activated under the action of chemical agents by inducing either rapid, nonsynchronous, and transient loss of transmembrane mitochondrial potential ({Delta}{Psi}m), followed by prompt recovery of mitochondrial integrity, or by an irreversible collapse of {Delta}{Psi}m accompanied by mitochondrial swelling (77) , the mode of dissipation of mitochondrial electrochemical potential occurring during homocysteine plus copper-induced apoptosis was defined by flow cytometry with the fluorescent probe DiOC6(3) (78) . With the use of fluorescent probes, similar fluorescence values are observed in cells with rapid and transient loss of {Delta}{Psi}m and in cells with stable {Delta}{Psi}m, whereas lower fluorescences are measured in cells with an irreversible collapse of {Delta}{Psi}m (77 , 79) . In our experimental conditions, the lower fluorescence values observed after treatment with Hcy plus Cu2+ (cells with low {Delta}{Psi}m) than in untreated cells support the fact that Hcy plus Cu2+ induces an irreversible collapse of {Delta}{Psi}m. This drop of {Delta}{Psi}m associated in our investigation with a translocation of cytochrome c into the cytosol could play an important role in the caspase activation cascade to further favor caspase-3 activation, PARP cleavage, and internucleosomal DNA fragmentation occurring in Hcy plus Cu2+-treated ECV304 cells. Indeed, we can suppose that cytochrome c associates with Apaf-1 in the presence of dATP or ATP and induces its oligomerization (58) . Furthermore, the oligomeric Apaf-1 complex would recognize the inactive procaspase-9, forming the ‘apoptosome’ (80) , which in turn would induce autocatalytic processing of procaspase-9. Subsequently, the mature caspase-9 would activate its primary downstream target, procaspase-3, which would cleave some nuclear proteins such as PARP, leading to a typical apoptotic DNA fragmentation in multiples of 180 to 200 bp (59) . As the efficiency of Hcy plus Cu2+ in inducing apoptosis was inversely correlated with the duration of culture before treatment, we attempted to define which cellular parameter(s) could vary with the time of culture and simultaneously counteract cell death.

There is some evidence that Hcy plus Cu2+ could initiate cytotoxic effects through the induction of an oxidative stress. In the presence of physiological concentrations of Cu2+, Hcy is oxidized, leading to the generation of superoxide anions and hydrogen peroxide (35) . Moreover, after addition of Hcy plus Cu2+ to cultured HeLa cells, the intracellular concentration of glutathione decreases, release of glutathione to the culture medium increases, and there is a lower proportion of intracellular reduced thiols (21) . Since Hcy represents a part of the redox thiol system, changes in extracellular or intracellular Hcy concentrations may lead to an altered redox thiol status that may be relevant for several cellular functions. As the tripeptide thiol glutathione plays a pivotal role in the control of cellular functions (39) and of apoptosis induced by numerous agents (38 , 40) , we examined the effects of Hcy plus Cu2+ on the ectoenzyme {gamma}-GT, which catalyzes the first step in the extracellular transpeptidation of glutathione via the {gamma}-glutamyl cycle into amino acid intermediates (39 , 41) . Indeed, the ability of various drugs to induce apoptosis in different tumoral cell lines has been shown to depend on {gamma}-GT activity. Thus, cells with high {gamma}-GT activity were often resistant to antitumoral drugs (42 , 43) .

In the present study, there was a close correlation between the period of culture before treatment and the enhancement of {gamma}-GT activity associated with a higher reduced GSH content per cell. There was also an inverse correlation between the enhancement of {gamma}-GT activity and the efficiency of Hcy plus Cu2+ to induce apoptosis. Taken together, these data led us to suppose that {gamma}-GT activity could constitute an additional mechanism of resistance of confluent cells (81) that could be involved in the control of Hcy plus Cu2+-induced apoptosis. This hypothesis was supported by the use of the glutamine antagonist acivicin, a specific competitive inhibitor of {gamma}-GT activity (46) and dexamethasone, which has (among its numerous effects) the ability to stimulate {gamma}-GT activity (47) . In the presence of acivicin, the efficiency of Hcy plus Cu2+ in inducing apoptosis was enhanced, whereas in the presence of dexamethasone the ability of Hcy plus Cu2+ in inducing apoptosis was reduced. As the use of acivicin and dexamethasone-reducing and -enhancing {gamma}-GT activity, respectively, was also able to modulate the cytotoxicity of antitumoral drugs such as daunorubicin, VP-16, vinblastine, or Ara-C, our data suggest that {gamma}-GT activity could constitute an additional important parameter in the control of chemical-induced apoptosis. This hypothesis is reinforced by data obtained from Ramos B cells that clearly demonstrate that the overexpression of {gamma}-GT obtained by transfection allows protection from apoptosis (82) .

In conclusion, Hcy taken individually has no cytotoxic effects on ECV304 cells or HUVECs even when it is used at the high concentrations corresponding to those found in the plasma of hyperhomocysteinemic patients. However, when associated with Cu2+ taken in a range of concentrations commonly found in human plasma, Hcy becomes strongly cytotoxic and involves (in both ECV304 and HUVECs) a process of cell death by apoptosis that is under the control of {gamma}-GT activity. In the present study, as the efficiency of some antitumoral drugs (daunorubicin, VP-16, vinblastine, or Ara-C) was also reported to depend on {gamma}-GT activity, it is tempting to speculate that {gamma}-GT could play an important role in the control of apoptosis induced by numerous chemical agents. Taken together, therefore, our data shed new insight on the cellular dysfunctions induced by Hcy and might allow the development of new therapies based on either the stimulation or inhibition of {gamma}-GT activity in order to counteract or stimulate cell death, respectively, depending on the pathology considered.


   ACKNOWLEDGMENTS
 
This work was supported by the Université de Bourgogne, the Conseil Régional de Bourgogne, the Institut National de la Santé et de la Recherche médicale (Inserm), the Fondation pour la Recherche Médicale, and the Fondation de France. The authors are indebted to Mr. Jonathan Ewing for reviewing the English version of the manuscript.

Received for publication December 8, 2000. Revision received April 27, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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