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Originally published In Press as doi:10.1074/jbc.M602500200 on July 20, 2006

J. Biol. Chem., Vol. 281, Issue 40, 29542-29557, October 6, 2006
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SLCO/OATP-like Transport of Glutathione in FasL-induced Apoptosis

GLUTATHIONE EFFLUX IS COUPLED TO AN ORGANIC ANION EXCHANGE AND IS NECESSARY FOR THE PROGRESSION OF THE EXECUTION PHASE OF APOPTOSIS*

Rodrigo Franco and John A. Cidlowski1

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, March 16, 2006 , and in revised form, June 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH]i, nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH]i can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH]i in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and {alpha}-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the execution phase of apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is a ubiquitous genetically encoded pathway that enables cells to undergo highly regulated death in response to specific signals. It has also been observed to occur as a consequence of distinct pathologies, and its deregulation can lead to autoimmune diseases, cancer, and neurodegenerative diseases (1). It is characterized by the activation of precise pathways leading to specific biochemical and morphological alterations, including changes in the intracellular ionic homeostasis, cell shrinkage, phosphatidylserine externalization, chromatin condensation, DNA degradation, membrane blebbing, and apoptotic body formation (1, 2). Fas ligand (FasL)2-induced apoptosis by binding to its receptor Fas (CD95/Apo-1) has been reported to play a critical role in immune homeostasis. Fas receptor-mediated apoptosis has also been implicated in the pathogenesis of fulminant hepatitis, postischemic neuronal degeneration, traumatic brain injury, and inflammatory lung diseases. Its deregulation is also associated with progression and metastasis of tumors and autoimmune lymphoproliferative syndrome (3).

GSH is the predominant low molecular weight thiol in animal cells. It participates in many cellular reactions, including antioxidant defense of the cell, drug detoxification, and cell signaling, and has been reported to be necessary for the proliferation of several cells, including lymphocytes (4–6). A reduction in the intracellular GSH concentration ([GSH]i) has been reported during cell death in response to different apoptotic stimuli, including death receptor-induced (7–10), stress-induced, (11), and drug-induced cell death (12). Several studies have shown a correlation between GSH efflux and the progression of apoptosis (13, 14), and a recent report suggests that inhibition of GSH release in apoptosis is able to rescue cells from apoptosis (8); however, the precise mechanisms involved in intracellular GSH (GSHi) loss have not been identified. Glutathione synthesis occurs intracellularly, and its catabolism occurs by a series of both enzymatic and plasma membrane export mechanisms. Glutathione degradation occurs extracellularly; therefore, the export of GSH and GSH adducts is also an important step in its turnover (15). To date, two GSH transporter families have been implicated in GSH efflux across the plasma membrane. These include the multidrug-resistant protein (MRP), encoded by ABCC genes, subfamily of the ATP binding cassette transporters (ABC transporters), and the organic anion-transporting polypeptide proteins (OATP), encoded by SLCO genes. During apoptosis, GSHi loss has been suggested to be mediated by activation of GSH active transport (7, 8, 11, 16); however, neither the identity of the transport mechanism involved in GSH extrusion nor its relationship with the progression of apoptosis have been elucidated.

In this work, we present evidence for the involvement of an organic anion exchange mechanism (a SLCO/OATP-like transporter) in the reduction of [GSH]i observed during FasL-induced apoptosis. Moreover, we show that the GSHi loss is a necessary step for the progression of the execution phase of the cell demise.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—RPMI 1640, penicillin/streptomycin, and heat-inactivated fetal calf serum were from Invitrogen. FasL was from Kamiya Biomedical Co. (Seattle, WA). MK571 was from BioMol (Plymouth, PA). 5-Carboxyfluorescein diacetate (CFDA), 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF), and monochlorobimane (mBCl) were from Molecular Probes, Inc. (Eugene, OR). The Cytofix/Cytoperm kit and the monoclonal antibodies PE-conjugated anti-active caspase-3, FITC-conjugated anti-PARP cleavage site-specific and FITC-conjugated anti-MRP1 monoclonal antibody were from BD Biosciences. Anti-caspase-3 and -7, anti-BID, anti-PARP, and anti-{alpha}-fodrin antibodies (monoclonal antibodies; human-specific) were from Cell Signaling Technology Inc. (Beverly, MA). All other reagents were from Sigma.

Cell Culture and Media—Jurkat cells, E6.1 clone (human leukemia) were obtained from American Tissue Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 31 mg/liter penicillin, and 50 mg/liter streptomycin at 37 °C in a 7% CO2 atmosphere. Jurkat cells (5–7 x 105 cells/ml) were incubated with FasL for the time indicated for the induction of apoptosis. In the medium containing high GSH, NaCl was substituted with 25 mM L-glutathione, maintaining the same osmolarity of the media. For choline- or N-methyl-D-glucamine (NMDG)-substituted medium, RPMI 1640 medium was initially made substituting NaCl for either choline chloride or NMDG. Additionally, choline bicarbonate and magnesium phosphate were used in place of sodium bicarbonate and sodium phosphate, respectively. Medium osmolarity was measured on a Wescor 5500 vapor pressure osmometer (Logan, UT).

Total Intracellular Glutathione Determinations, [glutathione]i—Quantitative, colorimetric determination of total intracellular glutathione concentration, [glutathione]i, was measured using a Bioxytech GSH-420 kit (Oxis Research, Portland, OR) following the manufacturer's specifications. This method is based on the formation of a chromophoric thione, using 4-chloro-1-methyl-7-trifluoromethylquinolinum methylsulfate as chromogen, whose absorbance was measured at 420 nm on a DU650 spectrophotometer (Beckman, Fullerton, CA). The absorbance at 420 nm is linearly proportional to the concentration of glutathione. A calibration curve with provided calibrator solutions was performed for each experiment. For each sample, 2 x 107 cells/sample were used, and total glutathione content was normalized to protein concentration for each sample. This method determines [glutathione]i (i.e. both reduced (GSH) and oxidized forms) (GSSG is reduced to GSH using tris(2-carboxyethyl)phosphine). Results are expressed as a percentage of change in the intracellular GSH content versus control conditions.

Reduced (GSH) and Oxidized (GSSG) Glutathione Determinations—Cells were incubated with 250 µM L-({alpha}S,5S)-{alpha}-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) for 4 h to inhibit catabolism of released GSH by the {gamma}-glutamyl-transferase. Then cells (2 x 107 cells/ml) were induced to undergo apoptosis with FasL in the presence of acivicin. Samples were centrifuged, and aliquots were taken for extracellular determinations. Pellets were resuspended in ice-cold phosphate-buffered saline (PBS), homogenized, and kept frozen until use for intracellular determinations. The extracellular and intracellular GSH and GSSG concentrations were measured enzymatically using the GSH/GSSG-412 kit (Oxis Research). Samples were prepared in 5% metaphosphoric acid to remove proteins, with or without 1-methyl-2-vinyl-pyridium trifluoromethane sulfonate, a GSH-specific scavenger. Ellman's reagent DTNB (5',5-dithiobis-2-nitrobenzoic acid) reacts with GSH to form a product with an absorption maximum at 412 nm. GSSG was determined using glutathione reductase and NADPH to reduce GSSG to GSH, followed by reaction with Ellman's reagent. Results are normalized to protein concentration for each sample.

Flow Cytometry Assay—FasL-induced apoptotic parameters were analyzed by flow cytometry using a BD LSR II flow cytometer and BD FACSDiva Software (BD Biosciences) for data analysis, unless otherwise stated. Cells were analyzed at a cell density of 5 x 105 cells/ml, and in all cases 10,000 cells were analyzed. Fluorophores were diluted in Me2SO and preloaded at 37 °C, 7% CO2. In all cases, the final concentration of Me2SO never exceeded 0.1%. When indicated, propidium iodide (PI) was added at a final concentration of 10 µg/ml to evaluate plasma membrane integrity. Sequential analysis of the distinct fluorophores was used, and, unless otherwise indicated, cells with increased PI fluorescence were excluded during the analysis. For PI, cells were excited with an argon 488-nm laser, and emission was acquired with a 695/40 filter. Histograms and plots in all cases are representative of at least three different experiments. Changes in different morphological and biochemical parameters during apoptosis induced by FasL in Jurkat cells were determined as follows.

Changes in Intracellular Glutathione Concentration, [GSH]i— For intracellular GSH (GSHi) measurements, cells were preloaded 10 min with 10 µM mBCl, which forms blue fluorescent adducts with intracellular glutathione (17, 18). Immediately prior to flow cytometry examination, PI was added. For mBCl, cells were excited with a UV 405 laser, and emission was acquired with a 440/40 filter. When indicated, mBCl fluorescence was plotted against changes in forward scatter, which was acquired by exciting the cells with an argon 488-nm laser.

5-Carboxyfluorescein (CF) Efflux Assay for Functional Assessment of Multidrug Resistance Protein Activity—Cells were incubated with 1 µM CFDA for 1 h at 37 °C then centrifuged and washed with CFDA-free medium. CFDA diffuses into cells, where it is cleaved by intracellular esterases, resulting in fluorescent CF, which is a substrate of ABCC/MRP transporters (19). Cells were then resuspended in CFDA-free medium and allowed to efflux CFDA for the time indicated at 37 °C in the presence or absence of ABCC/MRP activity reversal agents. Immediately prior to flow cytometry examination, PI was added. For CF fluorescence, samples were excited using an argon 488 laser with a 530/30 filter.

CDF Uptake Assay, for Functional Assessment of Organic Anion Transport Activity—Organic anion transport activity during apoptosis was evaluated by a CDF uptake assay. Organic anion transporters have previously been reported to transport organic anion fluorescein derivates, including CDF (19, 20). Apoptosis was induced in the presence of 5 µM CDF for the time indicated. Cells were then centrifuged, washed, and resuspended in PBS. For simultaneous analysis of [GSH]i levels, cells were incubated for an additional 10 min with mBCl as described previously. Immediately prior to flow cytometry examination, PI was added. For CDF fluorescence, samples were excited using an argon 488 laser with a 530/30 filter. An increase in organic anion transport (Formula) is reflected by CDF accumulation in cells as measured by an increase in fluorescence.

Jurkat Cell Immunolabeling Assay—Samples were washed once with ice-cold PBS and then fixed and permeabilized using the Cytofix/Cytoperm kit for 30 min at room temperature in dark according to the manufacturer's specifications. Cells were then centrifuged and washed with the Perm/Wash buffer and stained with PE-conjugated anti-active caspase-3, FITC-conjugated anti-PARP cleavage or FITC-conjugated rabbit anti-MRP1 for 1 h at room temperature in the dark. Following incubation, cells were washed with the PermWash buffer and analyzed by flow cytometry. PE and FITC fluorescence were detected using an argon 488 laser with 575/26 and 530/30 filters, respectively.

DNA Degradation Analysis by Flow Cytometry—Samples were pelleted and washed in ice-cold PBS. Then cells were fixed by the addition of 70% cold ethanol to a volume of ~1.5 ml, adjusted to 5 ml with cold 70% ethanol, and stored at –20 °C overnight. Fixed cells were pelleted, washed once in PBS, and stained in 1 ml of 20 µg/ml PI, 1 mg/ml RNase, in PBS for 20 min at room temperature. 7500 cells were analyzed by flow cytometry using a BD Biosciences FACSort flow cytometer and BD Cell Quest Software (BD Biosciences) for analysis. Region gates were set on a PI area versus width dot plot to exclude cell debris and cell aggregates.

Determination of mRNA Levels of ABCC/MRP and SLCO/OATP Genes by Real Time PCR—RNA extraction from Jurkat cells was performed using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's specifications. Reverse transcription of RNA was performed in PCR buffer containing 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 5.5 mM MgCl2, with murine leukemia virus reverse transcriptase, dNTP (deoxy-CTP, -GTP, -TTP, and -ATP), oligo(dT)16, and RNase inhibitor, all reagents from Applied Biosystems Inc. (Foster City, CA). The reverse transcription reaction was carried out at 25 °C for 9.5 min, 48 °C for 30 min, and 95 °C for 5 min. mRNA levels for the organic anion-transporting polypeptides (SLCO/OATP proteins) and the multidrug resistance protein (ABCC/MRP) members were determined by real time PCR on an Applied Biosystems Prism 7700 sequence detection system and Taqman gene expression assay (Assays-on-DemandTM;Applied Biosystems) according to the manufacturers' guidelines. Primer probes for the ABCC/MRP were as follows: ABCC1/MRP1 (transcript accession no. NM_019902 [GenBank] .1), ABCC2/MRP2 (NM_000392 [GenBank] .1), ABCC3/MRP3 (NM_020037.1), ABCC4/MRP4 (NM_005845 [GenBank] .2), ABCC5/MRP5 (NM_005688 [GenBank] .2), ABCC6/MRP6 (NM_001171 [GenBank] .2), ABCC7/MRP7 (NM_000492 [GenBank] .2), ABCC8/MRP8 (NM_000352 [GenBank] .2), ABCC9/MRP9 (NM_020298 [GenBank] .1). Primer probes for the SLCO/OATP transporters were as follows: SLCO1A2/OATP-A (NM_021094 [GenBank] .2), SLCO1B1/OATP-C (NM_006446 [GenBank] .2), SLCO1B3/OATP-8 (NM_019844 [GenBank] .1), SLCO1C1/OATP-F (NM_017435 [GenBank] .2), SLCO2A1/hPGT (NM_005630 [GenBank] .1), SLCO2B1/OATP-B (NM_007256 [GenBank] .2), SLCO3A1/OATP-D (NM_013272 [GenBank] .2), SLCO4A1/OATP-E (NM_016354 [GenBank] .3), SLCO4C1/OATP-H (NM_180991 [GenBank] .4), SLCO5A1/OATP-J (NM_030958 [GenBank] .1), and SLCO6A1/OATP-1 (NM_173488 [GenBank] .2), ordered as kits from Applied Biosystems. All probes were labeled with FAM TaqManTM fluorogenic probe (5'-6FAM-TGGCCCCAGCATGCGACCTC-tetra-methylrhodamine-3'). As reported, during PCR amplification, 5' nucleolytic activity of Taq polymerase cleaves the probe separating the 5' reporter fluorescent dye from the 3' quencher dye. This ends the activity of the quencher dye, and the reporter dye emits fluorescence, which increases in each cycle proportional to the rate of probe cleavage in real time. PCRs were carried out in 96-well plates (MicroAmp Optical 96-well reaction plate and optical caps; Applied Biosystems) in a 50-µl reaction mix volume containing the corresponding concentration of cDNA sample, 2.5 µl of primer probes, and 1x Taqman universal PCR master mix without AmpEraseUNG (containing MgCl2, dUTP, dATP, dCTP, dGTP, and Taq Gold polymerase; Applied Biosystems). Samples were denatured at 95 °C for 10 min and then subjected to 40 cycles of PCR (15 s at 95 °C to activate the TaqDNA polymerase and 1 min at 60 °C to anneal/extend).

Relative mRNA levels for ABCC/MRP and SLCO/OATP transporters were analyzed using a standard curve from 50 to 200 ng (five concentrations) of cDNA input to obtain the threshold cycle, CT. This CT value is determined as the cycle number at which the reporter fluorescent emission increases above a threshold level (usually 10 times the S.D. value of the base line) at a concentration of 50 ng of cDNA input. As control, we used GAPDH to correct for potential variation in RNA loading of amplification efficiency in each experiment. Data are expressed as the intercept CT values of each gene obtained from standard curves normalized against the GAPDH CT value (CT GAPDH/CT). All samples were amplified simultaneously in triplicate in one assay run. For amplification efficiency validation, standard curve equation values are shown in Table 1 for each gene analyzed (i.e. each primer/probe set used). Table 1 shows the standard curve equation for each gene, where the logarithm of each cDNA input concentration is plotted on the x axis, and the corresponding CT value is plotted on the y axis. R2 is calculated from the standard curve of five data pairs (0–200 ng). CT values greater than 40 indicate that mRNA levels were on the lower limit of detection and are not considered significant.


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  		TABLE 1
Gene expression of GSH efflux pumps in Jurkat cells

Messenger RNA levels were analyzed for the human multidrug resistance proteins (ABCC/MRP) and for the organic anion-transporting polyptide (SLCO/OATP) genes. Relative mRNA levels for ABCC/MRP and SLCO/OATP transporters were analyzed using a relative standard curve from 50–200 ng (five concentrations) of cDNA input to obtain the threshold cycle, CT. As control, we used GAPDH to correct for potential variation in RNA loading and efficiency of the amplification in each experiment. Data are expressed as the intercept CT values (ICT) of each gene obtained from standard curves normalized against the CT value for GAPDH (i.e. CT GAPDH/CT). The table shows the standard curve equation for each gene where the logarithm of each cDNA input concentration is plotted on the x axis and the corresponding CT value on the y axis. R2 is calculated from the standard curve of five data pairs (0–200 ng). CT of >40 or CT GAPDH/CT values of <0.6 indicate that mRNA levels were on the lower limit of detection. ND, no fluorescent emission signal was detected after 40 cycles. Data are means of three experiments ± S.E.

 


Figure 1
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  		FIGURE 1.
Changes in total intracellular GSH concentration [glutathione]i induced by FasL in Jurkat cells. Jurkat cells were incubated with FasL for the time indicated for the induction of apoptosis. A, quantitative biochemical (colorimetric) determination of changes in total intracellular glutathione induced by FasL. Left, kinetic curve of the changes in the total intracellular glutathione content, [glutathione]i, both GSH and GSSG, induced by 10 ng/ml FasL. Right, dose-response curve of the changes in [glutathione]i measured at 4 h in response to increasing FasL concentrations. Curves were fitted to exponential decay functions (r2 > 0.9). Data are means ± S.E. of n = 4. B–D, analysis of changes in the intracellular glutathione content GSHi by flow cytometry. B, effect of 1 mM diethylmalate (DEM) and 200 µM BSO on the mBCl fluorescence of Jurkat cells (0h). C, effect of 10 ng/ml FasL treatment for 4 h on the GSHi of Jurkat cells. Frequency histograms in B and C show the distribution of cells with different GSHi (i.e. different levels of mBCl fluorescence) (right of the black line). A reduction in GSHi is reflected by either the reduction in the mean mBCl fluorescence of the population of cells (B) or the appearance of a population of cells with reduced mBCl fluorescence (C)(left of the gray line). Plots are representative of at least four independent experiments. D, kinetic curves (right) and dose-response curves (left) of the decrease in the population of cells with high GSHi (i.e. high mBCl fluorescence) in response to different concentrations of FasL. Data in C were adjusted to exponential decay functions (r2 > 0.9) and are means ± S.E. of n = 4.

 
Protein Extraction and Western Immunoblotting—Cells were pelleted, washed once with ice-cold PBS, and lysed in buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100 and protease inhibitors (Complete Mini protease mixture; Roche Applied Science). Samples were sonicated and centrifuged, and the pellets were discarded. Then samples were assayed in a Beckman DU650 spectrophotometer for protein concentration using a Bio-Rad protein assay, and cell extracts were normalized to equal protein concentration. Loading buffer containing glycerol, SDS, and bromphenol blue was added, and samples were denatured at 99 °C for 5 min. Protein extracts, 50 µg/sample, were separated by SDS-PAGE on 4–20% gradient polyacrylamide Tris/glycine gels (Novex; Invitrogen) and transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline containing 0.05% Tween and 10% nonfat dry milk for 1 h. Antibodies were diluted in Tris-buffered saline containing 0.05% Tween and 5% nonfat dry milk. Blots were incubated with the corresponding primary antibody (1:1000) overnight. Then blots were incubated for 1 h with the corresponding horseradish peroxidase-linked secondary antibodies (Amersham Biosciences) diluted 1:10,000 in Tris-buffered saline containing 0.05% Tween and 0.5% nonfat dry milk. Blots were then visualized on film with the ECL chemiluminescent system (Amersham Biosciences). Blots were subsequently stripped using 65 mM Tris-HCl, pH 6.7, 100 mM beta-mercaptoethanol, 2% SDS buffer and reprobed for {alpha}-tubulin to verify equal protein loading.

Statistical Analysis—When indicated, significances of difference in mean values were calculated using the two-tailed Student's t test. The number of experiments is indicated in each case in the legends of the figures.


Figure 2
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  		FIGURE 2.
Changes in GSHi occur in two stages, which do not reflect loss of membrane integrity. Intracellular GSH measurements were performed by flow cytometry (A). Effect of 4-h treatment with 10 ng/ml FasL on the GSHi of Jurkat cells. Data are represented as contour plots of mBCl versus forward scatter. Populations were gated according to their GSHi levels on an mBCl fluorescence versus forward scatter plot: normal cells with high GSHi (population a depicted with a continuous line); and cells with reduced GSHi levels (b and c populations depicted with white dashed lines). B, changes in GSHi in response to different concentrations of FasL. Populations were gated as depicted in A, and represented individually. Black, population a; gray, population b; light gray, population c. Top, histograms of changes in GSHi in response to FasL treatment. Bottom, contour plots of PI fluorescence against mBCl fluorescence. Plots are representative of at least four independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FasL-induced Reduction in Total Intracellular Glutathione Concentration [GSH]i—A reduction in [GSH]i is a feature observed in apoptosis triggered by different stimuli, but the mechanisms involved in GSHi loss are not clear (21). Apoptotic agents have also been reported to stimulate the generation of reactive oxygen species, particularly hydrogen peroxide (22, 23), which might reduce [GSH]i by its oxidation to GSSG. However, several reports have demonstrated that the activation of an efflux transport mechanism for GSH extrusion rather than its oxidation to GSSG underlies the decrease in [GSH]i (7, 8, 11, 16). We first evaluated whether the reduction in [GSH]i was associated with a decrease in the total concentration of GSH. Changes in total glutathione concentration [glutathione]i of Jurkat cells induced to die by FasL were analyzed by a quantitative biochemical assay that measures both GSH and GSSG. If changes in [GSH]i reflect its oxidation, then [glutathione]i should not change. Treatment of Jurkat cells with FasL resulted in a time-dependent (Fig. 1A, left) and dose-dependent (Fig. 1A, right) reduction in [glutathione]i. These results support the hypothesis that activation of an efflux transport mechanism underlies the decrease in [GSH]i.

Apoptosis is a stochastic phenomenon that can be explained by exponential kinetics, which suggests first that the risk of cell death is constant, second that cell death occurs randomly in time, and third that the death of one cell is independent of that of other cells (24). Accordingly, kinetic and dose-dependent curves of the reduction in [GSH]i induced by FasL (Fig. 1A) fit well with exponential decay curves (r2 > 0.9), suggesting that the loss of GSHi correlates with the stochastic progression of cell death in Jurkat cells stimulated by FasL. To determine if the reduction in intracellular concentration occurs stochastically during FasL-induced apoptosis, we studied the reduction in [GSH]i by single cell analysis by flow cytometry. We simultaneously used the GSHi fluorophore mBCl and PI to evaluate for loss of membrane integrity. Histograms of mBCl fluorescence show that cells preloaded with the GSH dye had a high level of fluorescence, depicted as a single population of cells, reflecting the basal levels of GSHi (Fig. 1B, 0h panels). The fluorescence of Jurkat cells preloaded with mBCl was homogeneously reduced after acute treatment with 1 mM diethylmalate and 200 µM DL-buthionine-(S,R)-sulfoximine (BSO), agents that induce GSHi depletion by inhibiting the {gamma}-glutamyl cysteine synthetase and by direct reduction of mitochondrial GSH, respectively. This treatment has been previously reported to efficiently deplete cells of glutathione from both cytosol and mitochondria (25). After 6 h of diethylmalate and BSO treatment, Jurkat cells showed a high reduction in the mBCl fluorescence, indicating a high depletion of GSHi. It is important to note that mBCl reacts with all intracellular thiols; however, glutathione accounts for ~90% of nonprotein thiol content in cells. Thus, we consider that changes in mBClfluorescence largely reflect changes in the [GSH]i of Jurkat cells, although some overestimation of GSHi may reflect mBCl interaction with protein-thiols.

Fas ligand treatment induced a reduction in the population of cells with high [GSH]i (Fig. 1C, right of the white line) and the concomitant appearance of a population of cells with decreased [GSH]i (Fig. 1C, left of the white line). The appearance of discrete populations with different GSHi levels corroborates the observation that GSHi loss occurs stochastically. In accordance with the kinetic profile observed for the reduction in total glutathione concentration by FasL (Fig. 1A), kinetic and dose-response curves of the reduction in the percentage of cells with normal [GSH]i (i.e. high mBCl fluorescence) also fitted well with exponential decay regressions (r2 > 0.9) (Fig. 1D). When the changes in the GSHi levels were analyzed on contour plots of mBCl fluorescence versus forward scatter (Fig. 2A, contour plots), we observed that FasL induced the appearance of two populations of cells with reduced [GSH]i, with differences in their forward scatter properties (b and c populations depicted in dashed white lines) compared with normal cells (population a, depicted in a continuous gray line). Thus, we next analyzed the changes in the [GSH]i of this three populations individually. The histograms in Fig. 2B (upper panels) show the changes in the three populations of cells with reduced [GSH]i, in response to increasing concentrations of FasL. Populations were gated as depicted in Fig. 2A (right) and plotted individually. An increase in the population of cells within the first stage of reduced [GSH]i (gray) and a comparable decrease in cells with high [GSH]i (black) was observed to be proportional to the concentration of FasL used. The population of cells within the second stage of GSHi loss (light gray) did not increase, suggesting that these cells are in the degradation phase of apoptosis, hence in constant demise. Contour plots of mBCl against PI fluorescence (Fig. 2B, lower panels) showed that these populations do not have an increase in PI fluorescence, even in the presence of high concentrations of FasL. Thus, none of the populations of cells with differences in their [GSH]i values reflect GSH leakage due to loss of membrane integrity.


Figure 3
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  		FIGURE 3.
Changes in total intracellular GSH induced by FasL are not related to synthesis inhibition and normal turnover. Changes in GSHi were determined by flow cytometry as in Fig. 2B. A, progressive reduction of the GSHi content of Jurkat cells in the presence of 200 µM BSO. Results are represented as the ratio of mBCl fluorescence of treated cells (t) against the mBCl fluorescence of control cells (c) (in arbitrary units, AU) at the time indicated and are means ± S.E. of n = 3. B, effect of 100 ng/ml FasL treatment for 4 h on the GSHi of Jurkat cells in the presence or absence of BSO (200 µM). Frequency histograms show the distribution of cells with different GSHi. For comparison, the gray line shows the mean mBCl fluorescence of control cells. Plots are representative of at least three independent experiments.

 
Normal turnover of intracellular GSH in the presence of BSO (200 µM) requires up to 24 h of treatment to deplete intracellular GSH (Fig. 3A). When a strong specific thiol-oxidant like diethylmalate is added to the medium, GSH depletion is accelerated as seen in control experiments in Fig. 1C. In contrast, GSH loss by FasL observes a dose-dependent component, and at saturating concentrations of it (100 ng/ml) depletes Jurkat cells from intracellular GSH within a half-life of 4 h (Figs. 1D and 3B). Treatment with BSO (4 h) marginally reduced the GSHi content of control cells (Fig. 3B). However, in the presence of BSO, FasL-induced GSH loss remained unaffected. Thus, it is unlikely that GSH depletion during FasL-induced apoptosis is mediated by synthesis inhibition and normal turnover.

The Multidrug Resistance Proteins (ABCC/MRP) Do Not Participate in the Reduction of [GSH]i Induced by FasL—To date, two different kinds of glutathione efflux transporters have been identified at the molecular level in mammalian cells (26–29). They include the multidrug resistance proteins ABCC/MRP and the members of the organic anion-transporting polypeptide proteins, SLCO/OATP. Thus, we sought to determine which of these transport proteins was involved in GSH efflux during FasL-induced apoptosis. Human leukemia cell lines have been reported to express high levels of MRP proteins, and previous studies have suggested that Jurkat cells express high levels of the multidrug resistance protein 1 (ABCC1/MRP-1) (30, 31), but the presence of other subtypes of ABCC/MRP transporters has not yet been assessed. By means of real time PCR, we were able to detect several members of the ABCC/MRP protein family, including ABCC1/MRP-1, ABCC2/MRP-2, ABCC4/MRP-4, and ABCC5/MRP-5 (Table 1). The ABCC6/MRP-6 and ABCC7/MRP-7 genes were observed at the detection limit of the system. We also analyzed protein expression levels of the ABCC/MRP transporters in Jurkat cells. Immunolabeling analysis of fixed Jurkat cells showed a uniform increased level of fluorescence for cells stained with an FITC-conjugated monoclonal antibody against an intracellular epitope of ABCC1/MRP-1, compared with nonstained cells (Fig. 4A), corroborating that the protein expression of ABCC1/MRP-1 in Jurkat cells is homogeneous. The functional expression of ABCC/MRP transporters was also supported by CF-release experiments. The CF release assay is widely used to study the activity of ABCC/MRP proteins (32). Multidrug resistance protein-mediated transport of organic substrates, like CF, has been reported to be coupled to the extrusion of GSH, and both are inhibited by a wide variety of drug reversal agents (15, 27–29, 32–34). We observed that preloaded Jurkat cells actively extrude CF (Fig. 4B), and that its release was significantly inhibited by the drug reversal agents probenecid and MK571, being most sensitive to MK571. This pharmacological profile is consistent with the participation of ABCC/MRP proteins in the extrusion of CF. Moreover, 50 µM BSO pretreatment (overnight) of Jurkat cells, which reduces [GSH]i without reducing cell viability (not shown), significantly reduced CF efflux (Fig. 4B). This result supports the role of GSHi as a necessary cofactor in ABCC/MRP-mediated CF extrusion. Carboxyfluorescein has also been shown to be extruded by other members of the ABC transporter family, particularly by the ABCA1 (P-glycoprotein), whose activity may account for the active extrusion of CF. However, we did not detect significant mRNA levels or an immunolabeling signal for ABCA1 in Jurkat cells (data not shown). We also studied the effect of MK571 and probenecid on GSH efflux induced by FasL. Intracellular GSH loss induced by FasL was stimulated by MK571 and probenecid (Fig. 4, C and D). These agents decreased the population of cells with high [GSH]i and also increased the second population of cells with a high depletion of GSHi, suggesting that ABCC/MRP proteins do not mediate GSHi loss induced by FasL.

FasL-induced GSH Efflux Is Mediated by a SLCO-like Transport in Jurkat Cells—Members of the SLCO/OATP transporter family have recently been suggested to function as bidirectional transporters of glutathione in exchange for an organic anion (OA–) substrate (15, 26, 29, 35–39). Thus, SLCO/OATP proteins can function as GSH exchangers, rather than cotransporters as is the case for MRP transporters. Glutathione efflux by these proteins has been reported to be trans-stimulated by extracellular OA– substrates (i.e. its transport is accelerated by the presence of extracellular substrates that increase the exchange transport rate). Thus, the observation that GSH efflux was stimulated by MK571 and probenecid, which can be considered as OA–, suggested the possible participation of members of the organic anion transport polypeptide proteins (SLCO/OATP) rather than ABCC/MRP proteins (29, 36, 39) in GSH efflux induced by FasL. Adachi et al. (40) have recently reported high levels of mRNA for the SLCO3A1/OATP-D member in different cancer cell types, including leukemia cell lines. Thus, we determined whether Jurkat cells express mRNA for the different family members of the SLCO/OATP transporters. We observed that Jurkat cells express different members of SLCO/OATP protein family (Table 1), including the SLCO3A1/OATP-D, SLCO4A1/OATP-E, and SLCO4C1/OATP-H isoforms. The signal for SLCO1A2/OATP-A, SLCO2A1/hPGT, SLCO2B1/OATP-B, and SLCO5A1/OATP-J was at the limit of the detection system.


Figure 4
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  		FIGURE 4.
FasL-induced GSHi loss is not mediated by the multidrug resistance protein transporters, ABCC/MRP. A, immunodetection of ABCC1/MRP-1-positive cells. Cells were stained with FITC-conjugated anti-MRP1 human monoclonal antibody and then analyzed by flow cytometry. Frequency histograms show the mean fluorescence of cells without (left) of with (right) anti-MRP1 staining. For comparison, the black line shows the mean background fluorescence of cells without staining. B, functional analysis for multidrug resistance protein activity by CF-efflux assay. Cells were incubated with 1 µM CFDA (control). Then cells were centrifuged, washed, and resuspended in CFDA-free medium for 4 h, with or without the presence of the MRP reversal agents, MK571 (50 µM) and probenecid (500 µM). When indicated, wells were pretreated with 50µM BSO (overnight) to reduce GSHi, and then a CF efflux assay was performed in the presence of BSO for 4 h. Active efflux of CF is reflected by the reduction in the mean CF fluorescence of the population of cells (left of the black line). C, effect of 50 µM MK571 and 500 µM probenecid on the loss of GSHi induced by 10 ng/ml FasL for 4 h. Changes in GSHi were determined by flow cytometry and plotted as in Fig. 2B. In all cases, plots are representative of at least four independent experiments. D, kinetic curves of the effect of MK571 (50 µM) on the percentage of cells with high GSHi (black population in C) in both treated and untreated cells with 10 ng/ml FasL. Data in D were adjusted to linear regressions in the case of controls with or without MK571 or exponential decay functions when cells were exposed to FasL (r2 > 0.9). Data are means ± S.E. of n = 4.

 
Fig. 5, A and B, shows that glutathione efflux induced by FasL was trans-stimulated by taurocholic acid, estrone sulfate, and bromosulfophthalein (BSP), known OA– substrates for SLCO/OATP proteins. Similar to the effects observed in the presence of MK571 and probenecid, these agents not only decreased the population of cells with high [GSH]i (black) but also increased the second population of cells with a high degree of [GSH]i depletion (light gray). Thus, our results suggest the participation of a SLCO/OATP-like transporter in GSH efflux. Inhibitors of the liver sinusoidal membrane GSH transporter, L-methionine and L-cystathionine did not affect the reduction in [GSH]i by FasL (Fig. 5B) (16).

Another characteristic of the GSH transport mediated by SLCO/OATP proteins is that the driving force for the net GSH export is determined by the electrochemical gradient of GSH across the plasma membrane (7, 15). Glutathione is 90% freely distributed in the cytosol, where it is present in concentrations up to 10 mM in animal cells, whereas in plasma, concentrations are ~0.01 mM, and in cell culture medium, they are around 0.003 mM. Moreover, because GSH is negatively charged at physiological pH, there is a large intracellular negative potential of –30 to –60 mV that facilitates its extrusion. Thus, increasing the extracellular GSH concentration decreases the driving force of GSH extrusion and reduces net GSH efflux mediated by SLCO/OATP transporters. It is important to mention that high extracellular GSH stimulates SLCO/OATP efflux of GSHi; however, net reduction of [GSH]i is prevented by its exchange for extracellular GSH. As shown in Fig. 5A, an isosmotic increase of 25 mM in the extracellular concentration of GSH prevented GSHi loss during FasL-induced apoptosis. These results were also corroborated biochemically by analyzing the changes in [glutathione]i. Fig. 5C shows that the reduction in the total intracellular glutathione content induced by FasL is enhanced by MK571. High extracellular GSH concentrations prevented not only the loss of [glutathione]i but also its stimulation by MK571. These data suggest that both the stimulation of GSH efflux by OA– and its inhibition by high GSH medium act on the same GSH transport mechanism activated during apoptosis. Together, these results point to the participation of SLCO/OATP proteins rather than ABCC/MRP transporters in GSHi loss induced during FasL-induced apoptosis.


Figure 5
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  		FIGURE 5.
FasL-induced GSH efflux is mediated by a SLCO/OATP-like transporter in Jurkat cells. Intracellular GSH measurements by flow cytometry (A and B) and quantitative biochemical determination of total intracellular glutathione (C) were performed as explained before. Intracellular GSH loss induced by 10 ng/ml FasL (4 h) was assessed in the presence or absence of the SLCO/OATP substrates taurocholic acid (2 mM), estrone sulfate (2 mM), bromosulfophthalein BSP (1 mM), L-methionine (5 mM), and L-cystathionine (5 mM) and high extracellular GSH concentration (25 mM substitution of NaCl). In all cases, plots are representative of at least four independent experiments. Data in B and C are expressed as in Fig. 1 and are means ± S.E. of n = 4. *, p < 0.001 against corresponding controls; {zeta}, p < 0.005 against FasL treatment.

 
We observed that glutathione depletion in Jurkat cells upon FasL stimulation was mediated by a reduction in its [glutathione]i. To clarify if glutathione was getting effluxed in its reduced (GSH) or oxidized (GSSG) form, we next analyzed changes in both extracellular and intracellular concentrations of GSH and GSSG. This was performed enzymatically using 5,5'-dithiobis-(2-nitrobenzoic acid) as chromogen (Ellman's reagent). Table 2 shows that FasL induced the accumulation of extracellular GSH, concomitant with a decrease in GSHi. In accordance with the hypothesis of a SLCO/OATP-like exchanger involved in GSH loss, MK571 enhanced both extracellular GSH accumulation and GSHi depletion. Intracellular GSHi depletion was prevented in the presence of high extracellular GSH medium. A small but significant accumulation of extracellular GSSG was also observed (Table 2), which was also stimulated by MK571. However, intracellular GSSG formation was not detected upon FasL stimuli. Thus, the increase in extracellular GSSG might be ascribed to extracellular GSH autoxidation after its extrusion.


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  		TABLE 2
Extracellular and intracellular determinations of reduced (GSH) and oxidized (GSSG) glutathione

Cells (2 x 107 cells/ml) were incubated (4 h) with acivicin (250 µM) and then, stimulated with FasL (100 ng/ml) for 2 h with or without MK571 (50 µM), in the presence or absence of high extracellular GSH concentrations (25 mM). Samples were centrifuged, and aliquots of the medium were taken to determine changes in extracellular levels of GSH and GSSG. Pellets were used to assess levels of intracellular GSH and GSSG concentrations. Results were normalized against protein concentrations of each sample. The extracellular and intracellular GSH and GSSG concentrations were measured enzymatically using the GSH/GSSG-412 kit (Oxis Research, Portland, OR) according to manufacturer's instructions. Samples were prepared in 5% metaphosphoric acid to remove proteins, with or without 1-methyl-2-vinyl-pyridium trifluoromethane sulfonate, a GSH-specific scavenger. Ellman's reagent (5',5-dithiobis-2-nitrobenzoic acid) reacts with GSH to form a product with an absorption maximum at 412 nm. GSSG was determined using glutathione reductase and NADPH to reduce GSSG to GSH, followed by reaction with Ellman's reagent. Results are normalized to protein concentration for each sample and are means of n = 5 ± S.E.

 
FasL-induced GSHi Loss Is Paralleled by the Activation of an Organic Anion Uptake Mechanism—T-lymphocytes have previously been reported to possess OA– transport mechanisms that have been suggested to differ from that mediated by ABCC/MRP proteins (41). However, its characteristics and physiological implications are still unclear. If GSHi loss induced by FasL was mediated by a SLCO/OATP-like transporter that functions as a GSH/OA– exchanger, then GSHi loss should be accompanied by an increase in Formula. Simultaneous analysis of changes in [GSH]i and CDF uptake (as an index of Formula) showed that FasL-induced GSHi loss was paralleled by an increase in Formula (Fig. 6A). We observed that the first population of cells with a reduction in [GSH]i showed a high degree of Formula activity. Additionally, we observed that in the presence of CDF, control cells exhibited a significant increase in fluorescence compared with cells in the absence of CDF (blank). This finding suggests that there is a basal Formula activity in the absence of FasL. Carboxydichlorofluorescein uptake in control cells was enhanced by the presence of MK571 (Fig. 6B). This observation can be explained by the action of MK571 on the inhibition of CDF release from cells mediated by ABCC/MRP proteins (Fig. 4B). In accordance with the observations for GSHi loss, Formula was stimulated by MK571 and inhibited by high extracellular GSH medium (Fig. 6B). Uptake of nonpermeable fluorescein derivates has been ascribed to the activation not only of SLCO/OATP transporters but also of the family of organic anion transporters, SLC22A/OAT (20, 42). Accordingly, fluorescein derivatives uptake by SLC22A/OAT transporters has been reported to be inhibited by p-aminohippuric acid and coupled to the Na+ gradient across the plasma membrane (42, 43). As shown in Fig. 6C, FasL-induced Formula was not affected either by the addition of p-aminohippuric or by Na+-substituted media, suggesting that it was not mediated by SLC22A/OAT transporters. These data support the hypothesis of a common mechanism for both GSH efflux and Formula, with similar characteristics of a SLCO/OATP-like transporter (GSH/OA– exchanger).

Glutathione Transport Modulates the Progression of the Execution Phase of Apoptosis—The role of GSHi loss in the progression of apoptosis has not been clearly elucidated. Thus, we analyzed the relationship between GSH transport and the progression of the apoptotic cascade. Fig. 7A shows that inhibition of GSH efflux by high extracellular GSH medium inhibited the cleavage of the execution caspases 3 and 7 during FasL-induced apoptosis. The cleavage of execution caspase substrates PARP and {alpha}-fodrin was also inhibited by the presence of high extracellular GSH medium (Fig. 7B). Moreover, trans-stimulation of the GSH efflux transport by MK571 also increased the degree of caspase-3 and -7 cleavage as well as the cleavage of their substrates, PARP and {alpha}-fodrin (Fig. 7, C and D). Cleavage of execution caspases and of their substrates was also shown to be stimulated by probenecid, taurocholic acid, and estrone sulfate (not shown), thus suggesting that stimulation of GSH/OA– exchanges enhances the execution phase of apoptosis. These observations were corroborated by single cell immunolabeling analysis by flow cytometry. FasL induced the appearance of a population of cells stained positive against cleaved anti-caspase-3 and anti-PARP antibodies, which was significantly reduced in the presence of high extracellular GSH (Fig. 8, A and C). In addition, the stimulatory effects of MK571 on the cleavage of caspases and their substrates, analyzed by both flow cytometry and Western immunoblotting, were fully prevented by the presence of high extracellular GSH (Fig. 8, A–D), supporting the idea that they are mediated by the loss of GSHi.

We finally investigated the effects of GSH transport modulation on FasL-induced DNA degradation in Jurkat cells. Fig. 9 shows frequency histograms of PI fluorescence, showing the DNA content of Jurkat cells. FasL induced the appearance of a subdiploid peak of DNA, indicating the occurrence of degraded DNA (left of the black line). In the presence of high extracellular GSH, DNA degradation was highly reduced. Trans-stimulation of GSH efflux by MK571 did not increase the number of cells with degraded DNA. However, the observation that high GSH conditions prevent FasL-induced DNA degradation supports the hypothesis of the necessary role of a reduction in [GSH]i for the activation of endonucleases. Together, these results suggest that GSH transport through a SLCO/OATP transporter is necessary for the progression of the execution phase of FasL-induced apoptosis.


Figure 6
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  		FIGURE 6.
FasL activates an organic anion uptake mechanism that parallels GSHi loss during apoptosis. Apoptosis was induced by FasL (4 h) in the presence or absence (blank) of 5 µM CDF. Cells were then washed and resuspended with PBS. Immediately prior to flow cytometry examination, PI was added. Intracellular GSH measurements were performed as explained before. Populations were gated and represented according to their GSHi as in Fig. 2A. Then individual populations were analyzed for CDF fluorescence, which reflects organic anion uptake, Formula. A, changes in GSHi and Formula in response to different concentrations of FasL. Data are represented as contour plots of mBCl versus CDF fluorescence (top) and histograms of changes in CDF fluorescence (bottom). B, effect of 50 µM MK571 and high extracellular GSH medium (+GSH) on Formula induced by FasL. Frequency histograms in A and B show the levels of CDF fluorescence for the population of cells gated according to their GSHi. An increase in Formula is reflected by an increase in the CDF fluorescence. For comparison, the gray line shows the mean fluorescence of control cells in the presence of CDF for 4 h. In all cases, plots are representative of at least three independent experiments. C, effect of the OAT inhibitor p-aminohippuric acid (PAH; 1 mM) and sodium (Na+)-substituted medium on Formula. Sodium-substituted medium was made by equimolar substitution of NaCl with choline chloride or N-methyl-D-glucamine (NMDG). Data are expressed as the percentage of cells with high CDF fluorescence (gray population) in each condition and are means ± S.E. of n = 4. *, p < 0.001 against corresponding controls.

 


Figure 7
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  		FIGURE 7.
Glutathione efflux is necessary for the cleavage and activation of the execution caspases. Apoptosis was induced by 10 ng/ml FasL for the time indicated in the presence or absence of high extracellular GSH medium (A and B) or 50 µM MK571 (C and D). Western blot analyses were done in whole cell lysates prepared after treatment of cells with 10 ng/ml FasL for 4 h. Blots were incubated with the corresponding antibodies and then stripped and reprobed for {alpha}-tubulin to verify equal protein loading. Blots are representative of at least three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Programmed cell death, or apoptosis, is a fundamental process implicated not only in pathophysiological states but also during the development and normal physiology of organisms (1). The signaling pathways involved in the progression and activation of apoptosis have been extensively studied and dissected. Despite this, recent reports have discovered new roles for changes in the intracellular milieu of cells during apoptosis, as determinants in the activation of the apoptotic machinery including caspases and endonucleases. For example, it has been reported that changes in the cytosolic environment of cells, like changes in the ionic homeostasis, pH, and redox potential, modulate the progression of the apoptotic program (22, 23, 44). A reduction in the intracellular concentration of GSH, [GSH]i, is a common feature during apoptosis, but the mechanisms involved in this phenomenon are still far from being understood. Here we report for the first time that the reduction in [GSH]i during FasL-induced apoptosis is mediated by the activation of an efflux mechanism with characteristics of a SLCO/OATP-like transport (a GSH/OA– exchanger). Moreover, we present evidence that GSH transport tightly modulates the progression of the execution phase of apoptosis.

The reduction in GSHi during apoptosis has previously been reported to be associated with the activation of an active efflux transport mechanism. Several studies have demonstrated that reduced glutathione can be recovered from the extracellular medium, suggesting that GSH efflux occurs in its reduced but not its oxidized (GSSG) form (7, 8, 11, 16), discarding the role or reactive oxygen species in the reduction of [GSH]i. In this way, we observed that the reduction in GSHi by FasL is due to a total reduction in the [glutathione]i that does not involve nonspecific leakage through plasma membrane. Moreover, we did not detect the formation of intracellular GSSG upon FasL stimulation, discarding the role of intracellular GSH oxidation in GSH depletion. Thus, our results support previous observations implicating the activation of a plasma membrane transport involved in the extrusion of GSH during apoptosis.


Figure 8
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  		FIGURE 8.
Glutathione efflux is necessary for MK571-induced stimulation of the execution caspases cleavage and activation. Apoptosis was induced by 10 ng/ml FasL for 4 h in the presence or absence of high extracellular GSH medium and/or 50 µM MK571. A and C, immnunolabeling detection of cleaved caspase-3 and PARP by single cell analysis using flow cytometry. Frequency histograms in control panels show the distribution of cells with background fluorescence for PE or FITC antibodies (black). A second population (gray) with increased fluorescence for PE or FITC indicates the cells that are positive for the antibodies specific for cleaved caspase-3 and PARP. B and D, Western blot analyses of caspase-3 and -7, PARP, and {alpha}-fodrin cleavage. Blots were incubated with the corresponding antibodies and then stripped and reproved for {alpha}-tubulin to verify equal protein loading. Blots are representative of at least three independent experiments.

 


Figure 9
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  		FIGURE 9.
Effects of glutathione transport modulation on FasL-induced DNA degradation. Apoptosis was induced by 10 ng/ml FasL for 4 h in the presence or absence of high extracellular GSH (+GSH) medium and/or 50 µM MK571. Cells were fixed and stained with PI for flow cytometric analysis of DNA content. The frequency histograms show the distribution of cells with normal DNA (to the right of the gray line) and subdiploid DNA content (left of the black line). Plots are representative of at least three independent experiments.

 
Two types of GSH efflux transporters have been identified at the molecular level. These include the ABCC/MRP and the SLCO/OATP families of organic anion transporters. Previous studies have suggested that ABCC/MRP transporters may be involved in GSH efflux during apoptosis (11, 45). Glutathione transport mediated by ABCC/MRP has been shown to be involved in the detoxification of the intracellular environment of the cell, and it is coupled to the extrusion of a wide variety of structurally unrelated organic molecules. Drug reversal agents of ABCC/MRP transport inhibit ABCC/MRP transport of organic molecules as well as GSH extrusion (15, 27–29, 32–34). We report that Jurkat cells functionally express ABCC/MRP proteins from which we detect mRNA for several members, particularly ABCC1/MRP-1, ABCC2/MRP-2, ABCC4/MRP-4, and ABCC5/MRP-5. Multidrug resistance protein transport activity was clearly inhibited by the drug reversal agents MK571 and probenecid. However, we observed that these agents stimulate rather than reduce GSH efflux during FasL-induced apoptosis. This effect was unrelated to differences in the expression of the ABCC/MRP proteins, because we observe a homogeneous expression of at least the ABCC1/MRP1 in all of the population of Jurkat cells. These results contrast with previous reports showing that probenecid inhibits GSH efflux activated by FasL in HepG2 hepatoma cells (7). Thus, at least in lymphoid cells, GSH extrusion during apoptosis does not seem to be mediated by the ABCC/MRP family of transporters.

The organic anion-transporting polypeptides (SLCO/OATP) form a superfamily of sodium-independent transport systems that mediate the transmembrane transport of a wide variety of endogenous and exogenous amphipathic organic compounds. Recent studies have shown that these transporters accept GSH as a substrate in exchange for an organic anion (29, 36, 39). We report here that Jurkat cells express mRNA for the SLCO3A1/OATP-D, SLCO4A1/OATP-E, and SLCO4C1/OATP-H genes. Pharmacological characterization of SLCO/OATP transport activity has led to the observation that GSH extrusion can be trans-stimulated by the presence of extracellular organic molecules, including probenecid, and that the major driving force of SLCO/OATP-mediated GSH efflux is the electrochemical gradient of GSH across the plasma membrane (15, 26, 29, 35–40). The observation that GSH efflux during FasL-induced apoptosis was trans-stimulated by the MRP inhibitors MK571 and probenecid prompted us to study the role of an SLCO/OATP-mediated transport in GSHi extrusion activated by FasL. We observed that FasL-induced GSH efflux was also trans-stimulated by the SLCO/OATP substrates taurocholic acid, estrone sulfate, and BSP, further supporting the involvement of a SLCO/OATP-like transport mechanism. Furthermore, reducing the electrochemical gradient of GSH by high extracellular GSH concentration prevented both GSHi loss induced by FasL and its trans-stimulation by MK571. Accordingly, accumulation of extracellular GSH was shown to be induced by FasL and to be stimulated by MK571. Extracellular GSH accumulation was fully prevented by high GSH medium. Together these data argue against the possibility of an ABCC/MRP-like transporter mediating the GSH efflux during FasL-induced apoptosis. It is important to mention that the SLCO/OATP substrates were not able to stimulate GSH efflux in the absence of FasL treatment, unless higher concentrations of at least 10-fold increase were used (not shown). This suggests that the effect of the organic substrates is on the GSH efflux mechanism activated during apoptosis and not through the activation of a secondary GSH transport.

We also show for the first time that FasL induces the activation of a Formula mechanism that parallels the loss of GSHi, which further supports the role of a GSH/OA– exchanger (SLCO/OATP-like transporter) in GSH depletion during apoptosis. This Formula mechanism seems to be tonically active, as evidenced by the observation that there is a significant uptake of CDF in Jurkat cells in the absence of FasL treatment. These results also suggested that FasL-induced apoptosis might induce an increase in the exchange rate of the SLCO/OATP transporter involved, for either the GSHi or for another organic substrate present in the extracellular medium. A more detailed study will be necessary to elucidate the exact molecular identity of the SLCO/OATP involved, the OA– involved in the GSH exchange process, and, more importantly, the mechanisms involved in its modulation during apoptosis. We cannot rule out the possibility that other mechanisms might be involved in GSHi loss during apoptosis. It is unlikely that GSH depletion during FasL-induced apoptosis is mediated by synthesis inhibition and normal turnover, since it was shown to take up to 24 h in the presence of BSO, a specific inhibitor of GSH synthesis. Additionally, FasL-induced GSH loss was not modified by BSO. A recent study has shown that glutamate-L-cysteine ligase, the rate-limiting enzyme in GSH biosynthesis, is a direct target of caspase-3 (46). This may represent an additional mechanism for preventing GSHi replenishment during apoptosis.

Although GSHi loss is a common feature during apoptosis, its relationship to the progression of the cell death program has not been clearly established. Interestingly, an apoptosis-resistant phenotype has been associated with high intracellular levels of GSH (13, 14, 47, 48). Previous studies have reported that GSHi loss directly modulates both the permeability transition pore formation, and the activation of caspase-3 (21, 49–53). Other reports have proposed that GSHi loss is preceded by the activation of execution caspases (7). A role of intracellular thiol depletion for the activation of endonucleases in apoptosis has also been previously suggested (54). We present evidence that GSH transport modulates the activation of the execution phase of apoptosis that includes activation of execution caspases and endonucleases. We also observed that GSHi loss in apoptosis seems to occur in two stages. These observations underscore the requirement of studying the role of GSHi loss at different stages throughout apoptosis and not as a single event occurring during the apoptotic signaling cascade. In conclusion, we propose for the first time that GSH efflux during apoptosis is mediated by a GSH/OA– exchange transport with characteristics of a SLCO/OATP-like transporter. This transport modulates the activation of the signaling machinery involved in the execution phase of FasL-induced apoptosis and the further progression of the cell demise.


   FOOTNOTES
 
* The research was supported by the Intramural Research Program of NIEHS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Laboratory of Signal Transduction, NIEHS, National Institutes of Health, P. O. Box 12233, 111 T. W. Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-1564; Fax: 919-541-1367; E-mail: cidlows1{at}mail.nih.gov.

2 The abbreviations used are: FasL, Fas ligand; GSHi, intracellular glutathione; [GSH]i, intracellular glutathione concentration; [glutathione]i, total glutathione concentration (both reduced GSH and oxidized GSSG); OA–, organic anion; Formula, organic anion uptake; CDF, 5 (and 6)-carboxy-2',7'-dichlorofluorescein; mBCl, monochlorobimane; PE, phycoerythrin; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PI, propidium iodide; CF, 5-Carboxyfluorescein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BSO, DL-buthionine-(S,R)-sulfoximine; PARP, poly-(ADP-ribose) polymerase; MRP, multidrug resistance protein; FAM, fluorescein; OATP, organic anion transporting polypeptides. Back


   ACKNOWLEDGMENTS
 
We acknowledge Dr. John Pritchard and Dr. David S. Miller for review of the manuscript. We thank Maria Sifre for technical support