Buthionine sulfoximine-induced cytostasis does not correlate with glutathione depletion YU-JIAN KANG AND M. DUANE ENGER Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011 Kang, Yu-Jian, and M. Duane Enger. Buthionine sulfoximine-induced cytostasis does not correlate with glutathione depletion. Am. J. Physiol. 262 (CeZZ PhysioZ. 31): Cl22C127, 1992.-The effects of L-buthionine-(S,R)-sulfoximine (BSO) on the proliferation of normal rat kidney fibroblasts (NRK49F) were determined and compared with the effects of BSO on cellular glutathione (GSH) content. The proliferation rate of exponentially growing NRK-49F cells was found to be slowed in 0.01 and 0.1 mM BSO and arrested in 1.0 and 10 mM BSO. There is no retardation in the proliferation of cells cultured in 0.001 mM BSO. However, varying BSO concentrations at and above 0.1 mM did not result in concordant differences in the rate and extent of GSH depletion. A dose-dependent effect of BSO on GSH levels was observed at BSO concentrations (0.01 mM. BSO was found also to inhibit epidermal growth factor (EGF)-induced DNA synthesis in NRK-49F cells arrested by serum deprivation in a dose-dependent pattern dissimilar to that of BSO-induced cellular GSH depletion. Removal of BSO allowed cells to resume proliferation. Further, growth-arresting BSO treatments were found to affect neither cell viability nor colony-forming efficiency. Addition of exogenous GSH or cysteine overcame BSO inhibition of EGF-induced DNA synthesis but not BSO depletion of cellular GSH levels. BSO was further found to inhibit the uptake of cysteine, cystine, and a-[ 1-14C] methylaminoisobutyric acid (MeAIB) by the EGF-stimulated quiescent cells in a dose-dependent fashion. The results presented here thus demonstrate that BSO inhibits the proliferation of NRK-49F cells. This effect, however, does not correlate with BSO-induced cellular GSH depletion and is not due to an overt toxic effect. BSO-reduced amino acid uptake may represent a possible mechanism by which BSO exerts its effect on cell growth. cell proliferation; growth ine-(S, R) -sulfoximine
inhibition;
NRK-49F
cells; L-buthion-
(BSO) is the most effective of several sulfoximines that inhibit y-glutamylcysteine synthetase, the enzyme that catalyzes the first step in glutathione (GSH) synthesis (11,13). It has been used extensively to facilitate studies on the metabolism and functions of GSH both in vivo and in vitro (2, 18, 19, 26, 29). As a potential cancer therapeutic sensitizer, BSO has been employed, with variable success in terms of antitumor effect, to deplete GSH in several experimental tumor models (6, IO, 24). In spite of the apparent usefulness of BSO in experimental and clinical approaches that benefit from its specific inhibition of GSH synthesis, potential problems with side effects associated with BSO treatment have been reported (6-8). Previous studies (20) showed, for example, that treatment with BSO inhibits proliferation of A549 human lung carcinoma cells. It is not known whether and to what extent this BSO response occurs in some other tumor or normal cells. In this study, the effects of BSO on the growth of normal rat kidney (NRK-49F) cells were determined.
L-BUTHIONINE-(s,R)-SULFOXIMINE
Cl22
0363-6143/92
$2.00 Copyright
The data obtained demonstrate that BSO inhibits proliferation and DNA synthesis in NRK-49F cells in a dose-dependent fashion. NRK-49F cells were found to be much more sensitive to the antiproliferative effects of BSO than are A549 cells. It is shown also that the antiproliferative effects of BSO neither correlate directly with effects on cellular GSH content nor reflect an overt toxic effect. MATERIALS
AND
METHODS
Materials Epidermal growth factor (EGF), glutathione (GSH), GSH disulfide reductase, NADPH, BSO, and 5,5’-dithiobis(2-nitrobenzoate) (DTNB) were obtained from Sigma Chemical (St. Louis, MO). cy-[l- 14C] methylaminoisobutyric acid (MeAIB), L[““Slcysteine, and L-[35S]cystine were from Du Pont-New England Nuclear (Boston, MA). Tritiated [“Hlthymidine was the product of Amersham (Arlington Heights, IL). McCoy’s 5A medium was purchased from GIBCO (Grand Island, NY), and calf serum was purchased from Hyclone (Logan, VT). ScintiVerse BD was from Fisher Scientific (Chicago, IL). All other chemicals were from either Sigma Chemical or Aldrich (Milwaukee, WI). Cell and Cell Culture Normal rat kidney fibroblasts (NRK-49F), obtained from the American Type Culture Collection (ATCC CRL 1570), were routinely grown in McCoy’s 5A medium supplemented with 10% calf serum, 2.2 g/l of sodium bicarbonat.e, and antibiotics at 37°C and pH 7.0-7.2 in a humidified atmosphere of 95% air5% COz. Stock cultures were passaged at 3-day intervals. Cells were removed from monolayer stock cultures with trypsinEDTA (0.05% trypsin, 0.53 mM EDTA-4Na), counted with a plated at Coulter counter (model ZB1, Coulter Electronics), 21,000 cells/dish in 35-mm tissue culture dishes or at 60,000 cells/dish in 60-mm tissue culture dishes, and incubated for growth or GSH determination, respectively. Some cells were plated at 21,000 cells/dish in 35-mm tissue culture dishes, incubated for 72 h in medium containing 10% calf serum, and growth arrested by changing to medium with 0.1% calf serum and incubating for 36 h. Growth stimulation was effected by directly adding EGF to cultures. Determination
of
Cell Proliferation
Proliferation of exponentially growing cells was followed by determining the total number of cells in each dish at 12- or 24 h intervals. At the end of each interval, the monolayer culture was washed with warmed, phosphate-buffered saline (PBS), trypsinized, and diluted with a ~10 volume of medium. The resulting cell suspension was aliquoted and further diluted with a X5 or X10 volume of Isoton II saline solution prior to determination of cell number in the Coulter counter. Because potential artifacts may be encountered in cell number counts with a Coulter counter, such as changes in cell size or volume due to treatments or stage of cell growth and debris from lysed
0 1992 the American
Physiological
Society
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BSO
AND
CELL
cells, the aperture current and amplification of the Coulter counter were adjusted to maintain average pulse height, an upper discriminator was not used, and values obtained were calibrated with hemacytometer measurements. The experimental protocols are as described in the text and in Figs. 1-5. Determination
of Total Cellular
GSH Levels
The DTNB-GSH disulfide reductase recycling assay (30) was used to determine total cellular glutathione content. Briefly, the cells were trypsinized, rinsed with cold PBS, centrifuged at 4OC, resuspended in cold 5% sulfosalicylic acid, mixed vigorously with a vortex apparatus, and recentrifuged. The supernatant was then assayed for total GSH by measuring the color change of DTNB at 412 nm in the presence of GSH disulfide reductase and NADPH. Total protein content was determined by using the Pierce BCA protein assay reagents (Rockford, IL) as described by Smith et al. (27). Determination
of Incorporation
of fH]Thymidine
into DNA
Growth-arrested cells were stimulated with 10 rig/ml EGF after a 36-h incubation in medium containing 0.1% calf serum. The cultures were then pulse-labeled with 1 &i/ml [“HIthymidine between 4 and 8 h, 10 and 14 h, and 22 and 26 h postEGF stimulation. Some cultures were exposed to BSO 12 h before EGF stimulation. At the end of each period, the labeled monolayer was washed 3 times with PBS, and the cells were detached with trypsin-EDTA. After suspension in PBS-bovine serum albumin (PBS-BSA, 0.1% BSA), an aliquot was diluted with Isoton II for determination of cell number. The remainder of the cell suspension was mixed with 20% trichloroacetic acid (TCA) to give a final TCA concentration of 10% and centrifuged after 10 min at 4°C. The precipitate was washed with 10% TCA and then with 5% potassium acetate in EtOH. After centrifugation, the final pellet was dissolved in 0.1 M NaOH, aliquots of which were mixed with ScintiVerse BD and counted in a liquid scintillation spectrometer. Measurement
of Cysteine, Cystine, and MeAIB
Uptake
Cells were washed twice with a transport buffer (Dulbecco’s PBS supplemented with 16 mM D-glucose) and then incubated with transport buffer containing 0.15 mM, 0.30 mM, or 0.10 mM cystine, cysteine, or MeAIB, respectively, and adequate radiolabeled amino acid to obtain statistically sufficient levels of counts. After 30-min incubation, the reaction was stopped by quickly rinsing the dishes with ~2 concentrated amino acid in ice-cold D-PBS. After two rinses with the transport buffer, the cells were then lysed with 0.1 M NaOH/O.l% sodium dodecyl sulfate (SDS), aliquots of which were diluted with ScintiVerse BD for scintillation counting and total protein determination. Measurement
Cl23
GROWTH
cells/ml by centrifugation and resuspension. One drop of the cell suspension was then added to one drop of 0.4% trypan blue on the open surface of a hemacytometer slide. After mixing with a Pasteur pipette and staining for 2 min, the stained cells and total cells were counted under a microscope. Because the processes of trypsinization, cell separation, centrifugation, and resuspension may cause loss of membrane integrity, the assay was also performed by adding the trypan blue solution directly into the monolayer after removing the culture medium and gently washing twice with warm PBS. Stained cells were then counted in different fields under the microscope. One-hundred cells were scored per field. Colony formation efficiency (WE). Cells grown in 35-mm tissue culture dishes with or without treatment with 1.0 mM BSO for 60 h were washed with warmed PBS, trypsinized, resuspended in fresh medium containing 10% calf serum, replated at 450-550 cells/dish in 60-mm dishes, and incubated at 37°C for 10 days. The colonies that formed were stained with methanol-crystal violet solution and counted with Biotran III (New Brunswick) colony counter. RESULT'S
BSO was found to reduce NRK-49F proliferation rates at concentrations above 0.001 mM. The rate of cell proliferation was slowed in 0.01 mM (data not shown) and in 0.1 mM BSO (Fig. 1). BSO at concentrations of 1.0 mM and 10 mM caused a more markedly reduced rate of cell proliferation. Cells exposed to these higher concentrations ceased to proliferate within 72 h after addition of BSO (Fig. 1). At the concentrations used here, BSO effectively decreases cellular GSH levels in NRK-49F cells. Although dose-dependent depletion of cellular GSH occurs at BSO concentrations below 0.01 mM, there is no significant difference in the rate and the extent of GSH depletion at BSO concentrations above 0.1 mM. As shown in Table 1, treatment for 12 h at BSO concentrations of 0.001, 0.01, 0.1, 1.0, and 10 mM depletes cellular GSH to 58.1, 200
1I I
/
150
/6
-I
x
/
/
of BSO Toxic Effect
Reversibility. To determine the reversibility of the BSO antiproliferative effect, exponentially growing cells were treated with 1 mM BSO for 60 h and then BSO was removed by changing the BSO-containing medium to fresh medium. The recovery of cell proliferation was then followed. Nonadherent cells. The fraction of nonadherent cells was determined by collecting the culture medium from each dish after gentle shaking and counting the cells with a Coulter counter after ~5 dilution with Isoton II solution. Cell viability. Trypan blue uptake was used to determine the viability of BSO-treated cells. The cells were detached by trypsinization. A cell suspension was then made by adding culture medium to the trypsinized cells and concentrated to lo6
I
I
I
0
24
48 Time
I
72
I
1
96
120
(Hours)
Fig. 1. Effect of L-buthionine-(S,R)-sulfoximine (BSO) on proliferation of NRK-49F cells. BSO was added 12 h following subculture, as indicated by arrow, at concentrations of 10 mM (A), 1 mM (m), and 0.1 mM (0). Control cultures (0) carried same volume of BSO vehicle. Data represent mean t SE values from triplicate samples for each treatment.
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Cl24
BSO
AND
CELL
GROWTH
Table 1. Effect of BSO on cellular GSH content in NRK-49F cells Time, Subculture
Treatment
BSO treatment
12 16 20 24 36 60 84 ND,
h Control
0 4 8 12 24 48 72
0.001
47.324.6 57.31k2.3 62.3t4.3 55.324.1 52.1t3.2 27.5t1.2 22.1t2.9
43.9kl.O 42.5S.3 32.121.8 31.6tl.6 11.9t0.2 9.7t1.3
GSH levels are expressed as nmol/mg protein and presented not detectable with Tietze’s assay (~0.1 nmol/mg protein).
as means
26.0,14.7,13.3, and 10.5% of the respective control value. Further depletion of cellular GSH content occurs with increasing time of treatment such that BSO concentrations > 0.1 mM result in depletion of cellular GSH to a level undetectable with Tietze’s assay after 24 h. As also shown in Table 1 and in accord with observations in other cells (16, 17, 20, 23), cellular GSH content in control cells changes as a function of subculture time. To investigate further the nature of BSO’s antiproliferative effects, exponentially growing NRK-49F cells were arrested (in GO) by serum deprivation, and the effect of BSO on EGF-induced DNA synthesis was determined. As shown in Fig. 2, growth-arrested cells increase significantly their rate of DNA synthesis upon EGF stimulation. Cells labeled lo- to 14-h post-EGF stimulation showed the greatest increase in [3H]thymidine incorporation. This result accords with earlier studies showing that cells reach a peak in the rate of DNA synthesis (as judged by [3H]thymidine incorporation) l2h post-EGF stimulation (21). BSO at concentrations of 0.1 and 1.0 mM significantly decreased EGF-stimulated DNA synthesis. As also summarized in Table 2, the dose response study showed that 1.0 mM BSO is close to the inhibitory concentration of 50% (I&) level and 10 mM T
0
ABCD
AB 6
12 Time
24
(Hours)
Fig. 2. Epidermal growth factor (EGF)-induced [“Hlthymidine incorporation in growth-arrested NRK-49F cells and effects of BSO on this process. A: control; B: 10 rig/ml EGF; C: 10 rig/ml EGF and 0.1 mM BSO; and D: 10 rig/ml EGF and 1 mM BSO. BSO treatment was begun 12 h prior to EGF stimulation. Cells were labeled with [3H]thymidine between 4 h and 8 h, 10 h and 14 h, and 22 h and 26 h post-EGF stimulation, respectively. Time points represent median time for each labeling period, and data represent mean t SE values from triplicate samples for each treatment.
(BSO),
mM
0.01
0.1
35.4t2.9 23.4~~0.8 14.4t0.8 8.lt0.5 1.9t0.2 ND
23.0t0.8 15.4-c-1.1 8.1t0.8 ND ND ND
-I- SE from
triplicate
samples.
1.0
10.0
25.6k1.3 12.9t0.8 7.3t0.5 ND ND ND BSO,
23.0t0.3 12.9t0.8 5.820.5 ND ND ND
L-buthionine-(S,R)-sulfoximine;
Table 2. BSO dose effects on 3H incorporation and GSH levels BSO,
mM
0 0.01 0.1 1.0 10.0
3H Incorporation, nmol/mg protein
loot1 86t2 68-1-l 51t5 6t3
GSH nmol/mg
Level, protein
22.0t2.4 1.9t0.3 ND ND ND
Data represent means t_ SE from triplicate samples. Experiments entailed 12 h treatment with BSO prior to EGF stimulation and labeling with [“Hlthymidine lo- to 14-h post-EGF stimulation. 3H incorporation expressed as % of EGF stimulated only. ND, not detectable (CO.1 nmol/mg).
BSO almost completely blocks EGF-stimulated DNA synthesis. Cellular GSH content in growth-arrested NRK-49F cells is -20% of the peak level in exponentially growing cells. Such arrested cells were treated with varying concentrations of BSO for 12 h prior to EGF stimulation. As shown in Table 2, BSO concentrations above 0.1 mM decrease cellular GSH content to a similar, undetectable level. BSO at the lower concentration of 0.01 mM was found to decrease cellular GSH content to -9% of control. The data presented in Table 2 indicate that the BSO dose responses for effects on proliferation and cellular GSH differ, suggesting that BSO may affect cellular processes other than those specifically involving GSH synthesis. To further explore the nature of the BSO antiproliferative effect, the effects of concurrent as well as pretreatment with BSO were determined as were the effects of exogenous GSH (Fig. 3). After serum-arrested cells were treated with 1 mM BSO for 12 h, the BSO was removed by changing the medium, and EGF was added immediately. After 12 h of EGF stimulation, the cellular GSH content remained at an undetectable level, but these cells showed much less inhibition in their DNA synthesis rate than did cells that were continuously treated with BSO. Further, when GSH was added to the medium at the same time as EGF, a dose-dependent antagonist effect of GSH on BSO-induced inhibition on DNA synthesis was observed. The cellular GSH content, however, was not affected by the GSH treatment, i.e., it remained at an undetectable level after 12 h of GSH treatment (data not shown). Although GSH itself is not transported into the cells,
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BSO IO
AND
1 -
CELL
T
-
-
Oa EGF (rig/ml) BSO (mM) GSH (mM) CysH (mM)
n 0 0 0 0
IO 0 0 0
0.01 0
0.1 0
10 1 0 0.01
0.1
Treatment
Fig. 3. Effects of exogenous glutathione (GSH) inhibition of DNA synthesis. BSO was added stimulation, and GSH and cysteine were added EGF. Cultures were then labeled with [“Hlthymidine 14 h post-EGF stimulation. *Indicates that cells with BSO for 12 h. At time of EGF stimulation subjected to medium change and then exposed then compared with that obtained from control subjected to medium change and EGF stimulation Data presented here are mean & SE values from each treatment. MeAlB
Cysteine
BSO
Concentration
or cysteine on BSO 12 h prior to EGF simultaneously with between 10 and were pretreated only the cultures were to EGF. Result was cultures, which were (data not shown). triplicate samples for
Cystine
Cl25
GROWTH
Because cells take up cysteine by A/ACS transport systems (5) and MeAIB has been used as a model compound for studying the A system, the general effect of BSO on this system was determined by measuring MeAIB uptake. As shown in Fig. 4, BSO inhibited uptake of MeAIB by the EGF-stimulated quiescent NRK-49F cells by 25%, 30%, and 35% at BSO concentrations of 0.01, 0.1, and 1.0 mM, respectively. Cells take up cystine by a different transport system. In many cell types, this system has been identified as the system (1). The effect of BSO on cystine uptake T’ was also measured (Fig. 3). BSO inhibited cystine uptake by 13%, 16%, and 33% at BSO concentrations of 0.01, 0.1, and 1.0 mM, respectively. To explore the possibility that the BSO antiproliferative effect reflects an overt toxic response in NRK-49F cells, recovery of cell proliferation after removal of BSO was followed, and at the same time the number of nonadherent cells in BSO-treated cultures was compared with that of the control cultures. As shown in Fig. 5, removal of 1 mM BSO after a 60-h treatment, at which time cell growth was completely arrested, allowed the cells to resume proliferation. Further, the number of nonadherent cells in treated cultures remained relatively constant until 84 h of BSO treatment. Although an increasing percentage of nonadherent cells in BSOtreated cultures was observed after this time, a decreasing percentage was also shown after removal of BSO. After treatment with 1 mM BSO for 60 h, the viability of the cells does not change as judged by trypan blue assay. The percentages of stained cells in BSO-treated
(mM)
Fig. 4. Effects of BSO on cw-[l-14C]methylaminoisobutyric acia (MeAIB), cysteine, or cystine uptake by EGF-stimulated serum arrested NRK-49F cells. Data represent mean t SE values from triplicate samples for each treatment.
GSH may be subjected to breakdown into its amino acid constituents by y-glutamyltranspeptidase, a cell membrane associated enzyme. Through its reaction, the amino acids produced from GSH degradation are transported into the cells (12). One of these amino acids is cysteine, an essential nutrient for cultured cells. If BSO inhibits cell proliferation by blocking cysteine uptake, then increasing concentrations of cysteine in the medium may overcome the BSO effect. As shown in Fig. 3, addition of cysteine to medium indeed blocked the BSO antiproliferative effect in a cysteine dose-dependent fashion. The effects of BSO on cysteine uptake are summarized in Fig. 4. At concentrations of 0.01, 0.1, and 1 mM, BSO inhibits cysteine uptake by EGF-stimulated quiescent NRK-49F cells 14%, 16%, and 20%, respectively.
Time
(Hours)
Fig. 5. Reversibility of BSO effect on cell proliferation. Cells were exposed to 1 mM BSO for 60 h beginning 12 h after subculture (as indicated by the arrow). BSO-containing medium was removed (as indicated by MC), monolayer was washed with warmed PBS twice, and fresh medium without BSO was added to culture (A). Proliferation of control (0) and cells treated continuously with BSO (0) followed. In bottom panel, changes in fraction of nonadherent cells in cultures are indicated. All data represent mean t SE values from triplicate samples for each treatment.
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Cl26
BSO
AND
CELL
cultures and control cultures are 3.1 t 0.8 and 3.2 t 0.4, respectively. When the trypan blue solution was directly added to the monolayer cultures, no stained cells were observed in either culture. Finally, BSO treatment does not affect colony-forming potential. As shown in Table 3, the average size of each colony as well as the CFE are similar in treated and control cell populations. DISCUSSION
Previous studies showed that treatment of human lung carcinoma-derived A549 cells with BSO concomitantly decreases cellular GSH and inhibits cell proliferation (20). The studies reported here demonstrate that treatment with BSO results in a decrease in cellular GSH content and in an inhibition of cell growth in NRK-49F cultures also. The BSO antiproliferative effect, however, does not in its dose dependency correlate with its effect on cellular GSH content. Although dose-dependent GSH depletion was observed at BSO concentrations at and below 0.01 mM, no significant difference in the rate and extent of GSH depletion was detected at BSO concentrations above 0.1 mM. As indicated by Clark et al. (6), BSO concentrations ~0.1 mM result in inhibitor-enzyme saturation in CHO cells. Based on the results presented here, this would appear to be the case also in NRK-49F cells. However, marked differences were apparent in the patterns of NRK-49F growth inhibition induced by BSO at concentrations of 0.1, 1.0, and 10 mM. As demonstrated in Fig. 1, a BSO concentration of 0.1 mM results in a reduced rate, but not in cessation, of cell proliferation. Treatment with 1 and 10 mM BSO resulted in an eventual plateau in cell number. This occurred earlier in 10 mM than in 1 mM BSO. There was also found to be no correlation of the dose dependency for the effects of BSO on DNA synthesis and on GSH content in EGFstimulated quiescent cells. That BSO-induced growth inhibition does not simply act through its effect on GSH synthesis is further revealed by the fact that there was a much reduced and almost no inhibitory effect of BSO on DNA synthesis when the serum-arrested cells were treated with 1 mM BSO 12 h prior to but not during EGF stimulation, even though the cellular GSH content was measured to be at a nondetectable level throughout the period of EGF stimulation in the absence of concurrent BSO treatment. An effect of BSO on cellular processes other than GSH synthesis is thus again suggested. Exogenous GSH, when added to the culture medium, overcomes the effect of BSO on EGF-induced DNA synthesis in a dose-dependent fashion. Although GSH itself cannot be transported into the cells, its constituent amino acids can, through the reaction of y-glutamylTable 3. Colony-forming efficiency (CFE) Control
Cells plated, cells/dish Colonies formed, colonies/dish Colony size, mm2/colony CFE, % Data represent Cells were treated
538tl4 147t12 1.02t0.09 27.3
BSO
Treated
491kl2 138t8 1.05t0.06 28.1
means t SE from 6 samples for each treatment. with 1 mM BSO for 60 h prior to CFE determination.
GROWTH
transpeptidase, be released and then be transported into the cells. The cells would also gain amino acids, and possibly resynthesize GSH, if the y-glutamylcysteine produced is transported into the cell. Cellular GSH content, however, was not increased by the addition of GSH to the culture. This result, together with the observation of BSO dose-dependent inhibition of cysteine, cystine, and MeAIB uptake by EGF-stimulated quiescent NRK49F cells, suggests that nutrient limitation rather than cellular GSH depletion may be (one of) the mechanism(s) by which BSO exerts its effect on cell proliferation. A BSO inhibitory effect on cystine uptake by A549 cells (4) and on transport of y-glutamyl amino acids was also observed in mouse kidneys (12). It also has been shown that growth inhibition occurs in murine L5178Y lymphoma cells cultured in cystine-deficient medium (3). It is therefore possible that BSO inhibition of amino acid uptake and, thereafter, of protein synthesis is the mechanism by which BSO inhibits cell proliferation. However, cellular GSH depletion may be indirectly related to inhibition of amino acid uptake. Depletion of cellular GSH results in reduced export of GSH, which in turn would cause a change in extracellular thiol/disulfide status and therefore cell membrane function. The fact that addition of GSH or cysteine alone restored proliferation in BSOtreated cells strengthens this possibility. Further study is thus needed to investigate the nature and consequences of the effects of BSO on amino acid transport. The BSO antiproliferative response does not reflect an overt toxic effect because removal of BSO allows BSO-arrested cells to resume proliferation (Fig. 5) and BSO treatment does not affect cell viability and colonyforming efficiency (Table 3). Although concordant dose responses for BSO effects on cell growth and GSH levels were not observed, it is not thereby established that there is not a causal relationship between cellular GSH depletion and cell growth inhibition. It is proven only that it is not a simple, direct relationship. Several studies have shown that intracellular GSH levels much lower than those usually found to exist in growing cells may be adequate to support cell proliferation in lymphocytes and 3T3 fibroblasts (9, 14, 15, 25). This lower GSH level may represent the mitochondrial pool. As reported by Meredith and Reed (22), the half-time of the mitochondrial GSH pool is much longer than the half-time of the cytoplasmic pool, 30 t 3 h vs. 2 t 0.1 h, in isolated rat hepatocytes, and the onset of cell injury by ethacrynic acid correlated with the depletion of mitochondrial GSH, whereas the cytosolic pool could be depleted without affecting cell viability. The BSO treatment presented in this study would affect little, if any, of the mitochondrial GSH pool prior to the onset of the antiproliferative effect, based on the length of BSO treatment and the fact that there is no effect of such BSO treatment on cell viability and CFE. It is thus suggested that the BSO antiproliferative effect does not follow depletion of a GSH pool necessary for maintaining cell viability. This study demonstrated also that NRK-49F cells are much more sensitive to BSO’s antiproliferative effects than are A549 cells. BSO at a concentration of 0.001 mM represents a level close to the threshold for growth in-
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BSO
AND
CELL
hibition in NRK-49F cells. The threshold level for A549 cells is, however, on the order of 1 mM, a concentration at which significant inhibition of NRK-49F cell proliferation is apparent. The delay times for cell cytostasis due to addition of 10 mM BSO are 36 h and 12 h in A549 and NRK-49F cells, respectively. BSO inhibits activation-dependent DNA synthesis in CD2 and CD3 antigen-stimulated T lymphocytes (28) in such fashion that dose-dependent inhibition of DNA synthesis is observed at BSO concentrations between 10B5 M and 10m3 M. However, treatment of these cells with 10m3 M BSO for 16 h was found to decrease cellular GSH content only from 0.70 t 0.26 to 0.63 t 0.14 nmol per lo6 cells. The effects of 10m4 M and 10m5 M BSO on cellular GSH levels were not reported. However, the data presented indicate a pronounced inhibition of mitogeninduced DNA synthesis in T lymphocytes treated with BSO in the absence of a significant reduction in GSH content. In this study, the effects of BSO on cellular GSH content and on cell proliferation were determined simultaneously. Analysis of such effects in both exponentially growing cells and EGF-stimulated growth-arrested cells strongly indicates that BSO-induced growth inhibition does not correlate with cellular GSH depletion.
GROWTH
11.
12.
13.
14.
15.
16.
17.
18.
19.
We thank Mary Nims for manuscript preparation. This work was supported by the National Institute of Environmental Health Sciences and by Iowa State University. Address for reprint requests: Y.-J. Kang, Dept. of Pharmacology, Univ. of North Dakota, School of Medicine, 501 N. Columbia Rd., Grand Forks, ND 58203.
20.
Received
22.
20 December
1990; accepted
in final
form
1 August
1991.
REFERENCES 1. Bannai, S. Transport of cystine and cysteine in mammalian cells. Biochim. Biophys. Acta 779: 289-306, 1984. 2. Biaglow, J. E., M. E. Varnes, E. P. Clark, and E. R. Epp. The role of thiols in cellular response to radiation and drugs. Radiat. Res. 95: 437-455, 1983. 3. Brodie, A. B., J. Potter, W. W. Ellis, M. C. Evenson, and D. Reed. Glutathione biosynthesis in murine L5178 Y lymphoma cells. Arch. Biochem. Biophys. 210: 437-444, 1981. 4. Brodie, A. E., and D. J. Reed. Buthionine sulfoximine inhibition of cystine uptake and glutathione biosynthesis in human lung carcinoma cells. Toxicol. Appl. Pharmacol. 677: 381-387, 1985. 5. Christensen, H. N. Exploiting amino acid structure to learn about membrane transport. Adu. Enzymol. ReZat. Areas Mol. Biol. 49: 41101,1979. 6. Clark, E. P., E. R. Epp, J. E. Biaglow, M. Morse-Gandro, and E. Zachgo. Glutathione depletion, radio-sensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine. Radiat. Res. 98: 370-380, 1984. 7. Dethlefsen, L. A., J. E. Biaglow, V. M. Peck, and D. N. Ridinger. Toxic effects of extended glutathione depletion by buthionine sulfoximine on murine mammary carcinoma cells. Int. J. Radiat. OncoZ. Biol. Phys. 12: 1157-1160, 1986. 8. Dethmers, J. K., and A. Meister. Glutathione export by human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation. Proc. Natl. Acad. Sci. USA 78: 7492-7496, 1981. 9. Fischman, C. M., M. C. Udey, M. Kurtz, and H. J. Wedner. Inhibition of lectin-induced lymphocyte activation by 2-cyclohexene-l-one: decreased intracellular glutathione inhibits an early event in the activation sequence. J. Immunol. 127: 2257-2262, 1981. 10. Green, J. A., D. T. Vistica, R. C. Young, T. C. Hamilton, A.
21.
23.
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26.
27.
28.
29.
30.
Cl27
M. Rogan, and R. F. 0~01s. Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion. Cancer Res. 44: 5427-5431,1984. Griffith, 0. W. Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J. Biol. Chem. 257: 13704-13712, 1982. Griffith, 0. W., R. J. Bridges, and A. Meister. Transport of y-glutamyl amino acids: role of glutathione and y-glutamyl transpeptidase. Proc. Natl. Acad. Sci. USA 76: 6319-6322, 1979. Griffith, 0. W., and A. Meister. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. BioZ. Chem. 254: 7558-7560, 1979. Hamilos, D. L., and H. J. Wedner. The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-l-one by nuclear size transformation. J. Immunol. 135: 2740-2747, 1985. Hamilos, D. L., P. Zelarney, and J. Mascali. Lymphocyte proliferation in glutathione-depleted lymphocytes: direct relationship between glutathione availability and the proliferative response. Immunopharmacology 18: 223-235,1989. Harris, J. W., and H. M. Patt. Non-protein sulfhydryl content and cell-cycle dynamics of Ehrlich ascites tumor. Exp. CeZZ Res. 56: 134-151,1969. Harris, J. W., and S. S. Teng. Sulfhydryl groups during the S phase: comparison of cells from Gl, plateau-phase Gl, and GO. J. CeZZ. Physiol. 81: 91-96, 1973. Kang, Y.-J., and M. D. Enger. Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells. Cell BioZ. ToxicoZ. 3: 347-360, 1987. Kang, Y.-J., and M. D. Enger. Glutathione is involved in the early cadmium cytotoxic response in human lung carcinoma cells. Toxicology 48: 93-101, 1988. Kang, Y.-J., and M. D. Enger. Glutathione content and growth in A549 human lung carcinoma cells. Exp. CeZZ Res. 187: 177-179, 1990. Kang, Y.-J., and M. D. Enger. Cadmium inhibits EGF-induced DNA synthesis but increases cellular glutathione levels in NRK49F cells. Toxicology 66: 325-333, 1991. Meredith, M. J., and D. J. Reed. Status of the mitochondrial pool of glutathione in the isolated hepatocyte. J. BioZ. Chem. 257: 3747-3753,1982. Ohara, H., and T. Terasima. Variations of cellular sulfhydryl content during cell cycle of HeLa cells and its correlation to cyclic change of x-ray sensitivity. Exp. Cell Res. 58: 182-185, 1969. Ozols, R. F., K. G. Louie, J. Plowman, B. C. Behrens, R. L. Fine, D. Dykes, and T. C. Hamilton. Enhanced melphalan cytotoxicity in human ovarian cancer in vitro and in tumor-bearing nude mice by buthionine sulfoximine depletion of glutathione. Biochem. Pharmacol. 36: 147-153,1987. Shaw, J. P., and J.-N. Chou. Elevation of intracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts. J. Cell. Physiol. 129: 193-198, 1986. Skapek, S. X., M. Colvin, 0. W. Griffith, G. B. Elion, D. D. Bigner, and H. S. Friedman. Enhanced melphalan cytotoxicity following buthionine sulfoximine-mediated glutathione depletion in human medulloblastoma xenograph in athymic mice. Cancer Res. 48: 2764-2767,1988. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85, 1985. Suthanthiran, M., M. E. Anderson, V. K. Sharma, and A. Meister. Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens. Proc. NatZ. Acad. Sci. USA 87: 3343-3347,199o. Suzkake, K., B. J. Petro, and D. T. Vistica. Dechlorination of L-phenylalanine mustard by sensitive and resistant tumor cells and its relationship to intracellular glutathione content. Biochem. Pharmacol. 32: 165-167,1983. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Anal. Biochem. 27: 502522, 1969.
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inhibition;
NRK-49F
cells; L-buthion-
(BSO) is the most effective of several sulfoximines that inhibit y-glutamylcysteine synthetase, the enzyme that catalyzes the first step in glutathione (GSH) synthesis (11,13). It has been used extensively to facilitate studies on the metabolism and functions of GSH both in vivo and in vitro (2, 18, 19, 26, 29). As a potential cancer therapeutic sensitizer, BSO has been employed, with variable success in terms of antitumor effect, to deplete GSH in several experimental tumor models (6, IO, 24). In spite of the apparent usefulness of BSO in experimental and clinical approaches that benefit from its specific inhibition of GSH synthesis, potential problems with side effects associated with BSO treatment have been reported (6-8). Previous studies (20) showed, for example, that treatment with BSO inhibits proliferation of A549 human lung carcinoma cells. It is not known whether and to what extent this BSO response occurs in some other tumor or normal cells. In this study, the effects of BSO on the growth of normal rat kidney (NRK-49F) cells were determined.
L-BUTHIONINE-(s,R)-SULFOXIMINE
Cl22
0363-6143/92
$2.00 Copyright
The data obtained demonstrate that BSO inhibits proliferation and DNA synthesis in NRK-49F cells in a dose-dependent fashion. NRK-49F cells were found to be much more sensitive to the antiproliferative effects of BSO than are A549 cells. It is shown also that the antiproliferative effects of BSO neither correlate directly with effects on cellular GSH content nor reflect an overt toxic effect. MATERIALS
AND
METHODS
Materials Epidermal growth factor (EGF), glutathione (GSH), GSH disulfide reductase, NADPH, BSO, and 5,5’-dithiobis(2-nitrobenzoate) (DTNB) were obtained from Sigma Chemical (St. Louis, MO). cy-[l- 14C] methylaminoisobutyric acid (MeAIB), L[““Slcysteine, and L-[35S]cystine were from Du Pont-New England Nuclear (Boston, MA). Tritiated [“Hlthymidine was the product of Amersham (Arlington Heights, IL). McCoy’s 5A medium was purchased from GIBCO (Grand Island, NY), and calf serum was purchased from Hyclone (Logan, VT). ScintiVerse BD was from Fisher Scientific (Chicago, IL). All other chemicals were from either Sigma Chemical or Aldrich (Milwaukee, WI). Cell and Cell Culture Normal rat kidney fibroblasts (NRK-49F), obtained from the American Type Culture Collection (ATCC CRL 1570), were routinely grown in McCoy’s 5A medium supplemented with 10% calf serum, 2.2 g/l of sodium bicarbonat.e, and antibiotics at 37°C and pH 7.0-7.2 in a humidified atmosphere of 95% air5% COz. Stock cultures were passaged at 3-day intervals. Cells were removed from monolayer stock cultures with trypsinEDTA (0.05% trypsin, 0.53 mM EDTA-4Na), counted with a plated at Coulter counter (model ZB1, Coulter Electronics), 21,000 cells/dish in 35-mm tissue culture dishes or at 60,000 cells/dish in 60-mm tissue culture dishes, and incubated for growth or GSH determination, respectively. Some cells were plated at 21,000 cells/dish in 35-mm tissue culture dishes, incubated for 72 h in medium containing 10% calf serum, and growth arrested by changing to medium with 0.1% calf serum and incubating for 36 h. Growth stimulation was effected by directly adding EGF to cultures. Determination
of
Cell Proliferation
Proliferation of exponentially growing cells was followed by determining the total number of cells in each dish at 12- or 24 h intervals. At the end of each interval, the monolayer culture was washed with warmed, phosphate-buffered saline (PBS), trypsinized, and diluted with a ~10 volume of medium. The resulting cell suspension was aliquoted and further diluted with a X5 or X10 volume of Isoton II saline solution prior to determination of cell number in the Coulter counter. Because potential artifacts may be encountered in cell number counts with a Coulter counter, such as changes in cell size or volume due to treatments or stage of cell growth and debris from lysed
0 1992 the American
Physiological
Society
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BSO
AND
CELL
cells, the aperture current and amplification of the Coulter counter were adjusted to maintain average pulse height, an upper discriminator was not used, and values obtained were calibrated with hemacytometer measurements. The experimental protocols are as described in the text and in Figs. 1-5. Determination
of Total Cellular
GSH Levels
The DTNB-GSH disulfide reductase recycling assay (30) was used to determine total cellular glutathione content. Briefly, the cells were trypsinized, rinsed with cold PBS, centrifuged at 4OC, resuspended in cold 5% sulfosalicylic acid, mixed vigorously with a vortex apparatus, and recentrifuged. The supernatant was then assayed for total GSH by measuring the color change of DTNB at 412 nm in the presence of GSH disulfide reductase and NADPH. Total protein content was determined by using the Pierce BCA protein assay reagents (Rockford, IL) as described by Smith et al. (27). Determination
of Incorporation
of fH]Thymidine
into DNA
Growth-arrested cells were stimulated with 10 rig/ml EGF after a 36-h incubation in medium containing 0.1% calf serum. The cultures were then pulse-labeled with 1 &i/ml [“HIthymidine between 4 and 8 h, 10 and 14 h, and 22 and 26 h postEGF stimulation. Some cultures were exposed to BSO 12 h before EGF stimulation. At the end of each period, the labeled monolayer was washed 3 times with PBS, and the cells were detached with trypsin-EDTA. After suspension in PBS-bovine serum albumin (PBS-BSA, 0.1% BSA), an aliquot was diluted with Isoton II for determination of cell number. The remainder of the cell suspension was mixed with 20% trichloroacetic acid (TCA) to give a final TCA concentration of 10% and centrifuged after 10 min at 4°C. The precipitate was washed with 10% TCA and then with 5% potassium acetate in EtOH. After centrifugation, the final pellet was dissolved in 0.1 M NaOH, aliquots of which were mixed with ScintiVerse BD and counted in a liquid scintillation spectrometer. Measurement
of Cysteine, Cystine, and MeAIB
Uptake
Cells were washed twice with a transport buffer (Dulbecco’s PBS supplemented with 16 mM D-glucose) and then incubated with transport buffer containing 0.15 mM, 0.30 mM, or 0.10 mM cystine, cysteine, or MeAIB, respectively, and adequate radiolabeled amino acid to obtain statistically sufficient levels of counts. After 30-min incubation, the reaction was stopped by quickly rinsing the dishes with ~2 concentrated amino acid in ice-cold D-PBS. After two rinses with the transport buffer, the cells were then lysed with 0.1 M NaOH/O.l% sodium dodecyl sulfate (SDS), aliquots of which were diluted with ScintiVerse BD for scintillation counting and total protein determination. Measurement
Cl23
GROWTH
cells/ml by centrifugation and resuspension. One drop of the cell suspension was then added to one drop of 0.4% trypan blue on the open surface of a hemacytometer slide. After mixing with a Pasteur pipette and staining for 2 min, the stained cells and total cells were counted under a microscope. Because the processes of trypsinization, cell separation, centrifugation, and resuspension may cause loss of membrane integrity, the assay was also performed by adding the trypan blue solution directly into the monolayer after removing the culture medium and gently washing twice with warm PBS. Stained cells were then counted in different fields under the microscope. One-hundred cells were scored per field. Colony formation efficiency (WE). Cells grown in 35-mm tissue culture dishes with or without treatment with 1.0 mM BSO for 60 h were washed with warmed PBS, trypsinized, resuspended in fresh medium containing 10% calf serum, replated at 450-550 cells/dish in 60-mm dishes, and incubated at 37°C for 10 days. The colonies that formed were stained with methanol-crystal violet solution and counted with Biotran III (New Brunswick) colony counter. RESULT'S
BSO was found to reduce NRK-49F proliferation rates at concentrations above 0.001 mM. The rate of cell proliferation was slowed in 0.01 mM (data not shown) and in 0.1 mM BSO (Fig. 1). BSO at concentrations of 1.0 mM and 10 mM caused a more markedly reduced rate of cell proliferation. Cells exposed to these higher concentrations ceased to proliferate within 72 h after addition of BSO (Fig. 1). At the concentrations used here, BSO effectively decreases cellular GSH levels in NRK-49F cells. Although dose-dependent depletion of cellular GSH occurs at BSO concentrations below 0.01 mM, there is no significant difference in the rate and the extent of GSH depletion at BSO concentrations above 0.1 mM. As shown in Table 1, treatment for 12 h at BSO concentrations of 0.001, 0.01, 0.1, 1.0, and 10 mM depletes cellular GSH to 58.1, 200
1I I
/
150
/6
-I
x
/
/
of BSO Toxic Effect
Reversibility. To determine the reversibility of the BSO antiproliferative effect, exponentially growing cells were treated with 1 mM BSO for 60 h and then BSO was removed by changing the BSO-containing medium to fresh medium. The recovery of cell proliferation was then followed. Nonadherent cells. The fraction of nonadherent cells was determined by collecting the culture medium from each dish after gentle shaking and counting the cells with a Coulter counter after ~5 dilution with Isoton II solution. Cell viability. Trypan blue uptake was used to determine the viability of BSO-treated cells. The cells were detached by trypsinization. A cell suspension was then made by adding culture medium to the trypsinized cells and concentrated to lo6
I
I
I
0
24
48 Time
I
72
I
1
96
120
(Hours)
Fig. 1. Effect of L-buthionine-(S,R)-sulfoximine (BSO) on proliferation of NRK-49F cells. BSO was added 12 h following subculture, as indicated by arrow, at concentrations of 10 mM (A), 1 mM (m), and 0.1 mM (0). Control cultures (0) carried same volume of BSO vehicle. Data represent mean t SE values from triplicate samples for each treatment.
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Cl24
BSO
AND
CELL
GROWTH
Table 1. Effect of BSO on cellular GSH content in NRK-49F cells Time, Subculture
Treatment
BSO treatment
12 16 20 24 36 60 84 ND,
h Control
0 4 8 12 24 48 72
0.001
47.324.6 57.31k2.3 62.3t4.3 55.324.1 52.1t3.2 27.5t1.2 22.1t2.9
43.9kl.O 42.5S.3 32.121.8 31.6tl.6 11.9t0.2 9.7t1.3
GSH levels are expressed as nmol/mg protein and presented not detectable with Tietze’s assay (~0.1 nmol/mg protein).
as means
26.0,14.7,13.3, and 10.5% of the respective control value. Further depletion of cellular GSH content occurs with increasing time of treatment such that BSO concentrations > 0.1 mM result in depletion of cellular GSH to a level undetectable with Tietze’s assay after 24 h. As also shown in Table 1 and in accord with observations in other cells (16, 17, 20, 23), cellular GSH content in control cells changes as a function of subculture time. To investigate further the nature of BSO’s antiproliferative effects, exponentially growing NRK-49F cells were arrested (in GO) by serum deprivation, and the effect of BSO on EGF-induced DNA synthesis was determined. As shown in Fig. 2, growth-arrested cells increase significantly their rate of DNA synthesis upon EGF stimulation. Cells labeled lo- to 14-h post-EGF stimulation showed the greatest increase in [3H]thymidine incorporation. This result accords with earlier studies showing that cells reach a peak in the rate of DNA synthesis (as judged by [3H]thymidine incorporation) l2h post-EGF stimulation (21). BSO at concentrations of 0.1 and 1.0 mM significantly decreased EGF-stimulated DNA synthesis. As also summarized in Table 2, the dose response study showed that 1.0 mM BSO is close to the inhibitory concentration of 50% (I&) level and 10 mM T
0
ABCD
AB 6
12 Time
24
(Hours)
Fig. 2. Epidermal growth factor (EGF)-induced [“Hlthymidine incorporation in growth-arrested NRK-49F cells and effects of BSO on this process. A: control; B: 10 rig/ml EGF; C: 10 rig/ml EGF and 0.1 mM BSO; and D: 10 rig/ml EGF and 1 mM BSO. BSO treatment was begun 12 h prior to EGF stimulation. Cells were labeled with [3H]thymidine between 4 h and 8 h, 10 h and 14 h, and 22 h and 26 h post-EGF stimulation, respectively. Time points represent median time for each labeling period, and data represent mean t SE values from triplicate samples for each treatment.
(BSO),
mM
0.01
0.1
35.4t2.9 23.4~~0.8 14.4t0.8 8.lt0.5 1.9t0.2 ND
23.0t0.8 15.4-c-1.1 8.1t0.8 ND ND ND
-I- SE from
triplicate
samples.
1.0
10.0
25.6k1.3 12.9t0.8 7.3t0.5 ND ND ND BSO,
23.0t0.3 12.9t0.8 5.820.5 ND ND ND
L-buthionine-(S,R)-sulfoximine;
Table 2. BSO dose effects on 3H incorporation and GSH levels BSO,
mM
0 0.01 0.1 1.0 10.0
3H Incorporation, nmol/mg protein
loot1 86t2 68-1-l 51t5 6t3
GSH nmol/mg
Level, protein
22.0t2.4 1.9t0.3 ND ND ND
Data represent means t_ SE from triplicate samples. Experiments entailed 12 h treatment with BSO prior to EGF stimulation and labeling with [“Hlthymidine lo- to 14-h post-EGF stimulation. 3H incorporation expressed as % of EGF stimulated only. ND, not detectable (CO.1 nmol/mg).
BSO almost completely blocks EGF-stimulated DNA synthesis. Cellular GSH content in growth-arrested NRK-49F cells is -20% of the peak level in exponentially growing cells. Such arrested cells were treated with varying concentrations of BSO for 12 h prior to EGF stimulation. As shown in Table 2, BSO concentrations above 0.1 mM decrease cellular GSH content to a similar, undetectable level. BSO at the lower concentration of 0.01 mM was found to decrease cellular GSH content to -9% of control. The data presented in Table 2 indicate that the BSO dose responses for effects on proliferation and cellular GSH differ, suggesting that BSO may affect cellular processes other than those specifically involving GSH synthesis. To further explore the nature of the BSO antiproliferative effect, the effects of concurrent as well as pretreatment with BSO were determined as were the effects of exogenous GSH (Fig. 3). After serum-arrested cells were treated with 1 mM BSO for 12 h, the BSO was removed by changing the medium, and EGF was added immediately. After 12 h of EGF stimulation, the cellular GSH content remained at an undetectable level, but these cells showed much less inhibition in their DNA synthesis rate than did cells that were continuously treated with BSO. Further, when GSH was added to the medium at the same time as EGF, a dose-dependent antagonist effect of GSH on BSO-induced inhibition on DNA synthesis was observed. The cellular GSH content, however, was not affected by the GSH treatment, i.e., it remained at an undetectable level after 12 h of GSH treatment (data not shown). Although GSH itself is not transported into the cells,
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BSO IO
AND
1 -
CELL
T
-
-
Oa EGF (rig/ml) BSO (mM) GSH (mM) CysH (mM)
n 0 0 0 0
IO 0 0 0
0.01 0
0.1 0
10 1 0 0.01
0.1
Treatment
Fig. 3. Effects of exogenous glutathione (GSH) inhibition of DNA synthesis. BSO was added stimulation, and GSH and cysteine were added EGF. Cultures were then labeled with [“Hlthymidine 14 h post-EGF stimulation. *Indicates that cells with BSO for 12 h. At time of EGF stimulation subjected to medium change and then exposed then compared with that obtained from control subjected to medium change and EGF stimulation Data presented here are mean & SE values from each treatment. MeAlB
Cysteine
BSO
Concentration
or cysteine on BSO 12 h prior to EGF simultaneously with between 10 and were pretreated only the cultures were to EGF. Result was cultures, which were (data not shown). triplicate samples for
Cystine
Cl25
GROWTH
Because cells take up cysteine by A/ACS transport systems (5) and MeAIB has been used as a model compound for studying the A system, the general effect of BSO on this system was determined by measuring MeAIB uptake. As shown in Fig. 4, BSO inhibited uptake of MeAIB by the EGF-stimulated quiescent NRK-49F cells by 25%, 30%, and 35% at BSO concentrations of 0.01, 0.1, and 1.0 mM, respectively. Cells take up cystine by a different transport system. In many cell types, this system has been identified as the system (1). The effect of BSO on cystine uptake T’ was also measured (Fig. 3). BSO inhibited cystine uptake by 13%, 16%, and 33% at BSO concentrations of 0.01, 0.1, and 1.0 mM, respectively. To explore the possibility that the BSO antiproliferative effect reflects an overt toxic response in NRK-49F cells, recovery of cell proliferation after removal of BSO was followed, and at the same time the number of nonadherent cells in BSO-treated cultures was compared with that of the control cultures. As shown in Fig. 5, removal of 1 mM BSO after a 60-h treatment, at which time cell growth was completely arrested, allowed the cells to resume proliferation. Further, the number of nonadherent cells in treated cultures remained relatively constant until 84 h of BSO treatment. Although an increasing percentage of nonadherent cells in BSOtreated cultures was observed after this time, a decreasing percentage was also shown after removal of BSO. After treatment with 1 mM BSO for 60 h, the viability of the cells does not change as judged by trypan blue assay. The percentages of stained cells in BSO-treated
(mM)
Fig. 4. Effects of BSO on cw-[l-14C]methylaminoisobutyric acia (MeAIB), cysteine, or cystine uptake by EGF-stimulated serum arrested NRK-49F cells. Data represent mean t SE values from triplicate samples for each treatment.
GSH may be subjected to breakdown into its amino acid constituents by y-glutamyltranspeptidase, a cell membrane associated enzyme. Through its reaction, the amino acids produced from GSH degradation are transported into the cells (12). One of these amino acids is cysteine, an essential nutrient for cultured cells. If BSO inhibits cell proliferation by blocking cysteine uptake, then increasing concentrations of cysteine in the medium may overcome the BSO effect. As shown in Fig. 3, addition of cysteine to medium indeed blocked the BSO antiproliferative effect in a cysteine dose-dependent fashion. The effects of BSO on cysteine uptake are summarized in Fig. 4. At concentrations of 0.01, 0.1, and 1 mM, BSO inhibits cysteine uptake by EGF-stimulated quiescent NRK-49F cells 14%, 16%, and 20%, respectively.
Time
(Hours)
Fig. 5. Reversibility of BSO effect on cell proliferation. Cells were exposed to 1 mM BSO for 60 h beginning 12 h after subculture (as indicated by the arrow). BSO-containing medium was removed (as indicated by MC), monolayer was washed with warmed PBS twice, and fresh medium without BSO was added to culture (A). Proliferation of control (0) and cells treated continuously with BSO (0) followed. In bottom panel, changes in fraction of nonadherent cells in cultures are indicated. All data represent mean t SE values from triplicate samples for each treatment.
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Cl26
BSO
AND
CELL
cultures and control cultures are 3.1 t 0.8 and 3.2 t 0.4, respectively. When the trypan blue solution was directly added to the monolayer cultures, no stained cells were observed in either culture. Finally, BSO treatment does not affect colony-forming potential. As shown in Table 3, the average size of each colony as well as the CFE are similar in treated and control cell populations. DISCUSSION
Previous studies showed that treatment of human lung carcinoma-derived A549 cells with BSO concomitantly decreases cellular GSH and inhibits cell proliferation (20). The studies reported here demonstrate that treatment with BSO results in a decrease in cellular GSH content and in an inhibition of cell growth in NRK-49F cultures also. The BSO antiproliferative effect, however, does not in its dose dependency correlate with its effect on cellular GSH content. Although dose-dependent GSH depletion was observed at BSO concentrations at and below 0.01 mM, no significant difference in the rate and extent of GSH depletion was detected at BSO concentrations above 0.1 mM. As indicated by Clark et al. (6), BSO concentrations ~0.1 mM result in inhibitor-enzyme saturation in CHO cells. Based on the results presented here, this would appear to be the case also in NRK-49F cells. However, marked differences were apparent in the patterns of NRK-49F growth inhibition induced by BSO at concentrations of 0.1, 1.0, and 10 mM. As demonstrated in Fig. 1, a BSO concentration of 0.1 mM results in a reduced rate, but not in cessation, of cell proliferation. Treatment with 1 and 10 mM BSO resulted in an eventual plateau in cell number. This occurred earlier in 10 mM than in 1 mM BSO. There was also found to be no correlation of the dose dependency for the effects of BSO on DNA synthesis and on GSH content in EGFstimulated quiescent cells. That BSO-induced growth inhibition does not simply act through its effect on GSH synthesis is further revealed by the fact that there was a much reduced and almost no inhibitory effect of BSO on DNA synthesis when the serum-arrested cells were treated with 1 mM BSO 12 h prior to but not during EGF stimulation, even though the cellular GSH content was measured to be at a nondetectable level throughout the period of EGF stimulation in the absence of concurrent BSO treatment. An effect of BSO on cellular processes other than GSH synthesis is thus again suggested. Exogenous GSH, when added to the culture medium, overcomes the effect of BSO on EGF-induced DNA synthesis in a dose-dependent fashion. Although GSH itself cannot be transported into the cells, its constituent amino acids can, through the reaction of y-glutamylTable 3. Colony-forming efficiency (CFE) Control
Cells plated, cells/dish Colonies formed, colonies/dish Colony size, mm2/colony CFE, % Data represent Cells were treated
538tl4 147t12 1.02t0.09 27.3
BSO
Treated
491kl2 138t8 1.05t0.06 28.1
means t SE from 6 samples for each treatment. with 1 mM BSO for 60 h prior to CFE determination.
GROWTH
transpeptidase, be released and then be transported into the cells. The cells would also gain amino acids, and possibly resynthesize GSH, if the y-glutamylcysteine produced is transported into the cell. Cellular GSH content, however, was not increased by the addition of GSH to the culture. This result, together with the observation of BSO dose-dependent inhibition of cysteine, cystine, and MeAIB uptake by EGF-stimulated quiescent NRK49F cells, suggests that nutrient limitation rather than cellular GSH depletion may be (one of) the mechanism(s) by which BSO exerts its effect on cell proliferation. A BSO inhibitory effect on cystine uptake by A549 cells (4) and on transport of y-glutamyl amino acids was also observed in mouse kidneys (12). It also has been shown that growth inhibition occurs in murine L5178Y lymphoma cells cultured in cystine-deficient medium (3). It is therefore possible that BSO inhibition of amino acid uptake and, thereafter, of protein synthesis is the mechanism by which BSO inhibits cell proliferation. However, cellular GSH depletion may be indirectly related to inhibition of amino acid uptake. Depletion of cellular GSH results in reduced export of GSH, which in turn would cause a change in extracellular thiol/disulfide status and therefore cell membrane function. The fact that addition of GSH or cysteine alone restored proliferation in BSOtreated cells strengthens this possibility. Further study is thus needed to investigate the nature and consequences of the effects of BSO on amino acid transport. The BSO antiproliferative response does not reflect an overt toxic effect because removal of BSO allows BSO-arrested cells to resume proliferation (Fig. 5) and BSO treatment does not affect cell viability and colonyforming efficiency (Table 3). Although concordant dose responses for BSO effects on cell growth and GSH levels were not observed, it is not thereby established that there is not a causal relationship between cellular GSH depletion and cell growth inhibition. It is proven only that it is not a simple, direct relationship. Several studies have shown that intracellular GSH levels much lower than those usually found to exist in growing cells may be adequate to support cell proliferation in lymphocytes and 3T3 fibroblasts (9, 14, 15, 25). This lower GSH level may represent the mitochondrial pool. As reported by Meredith and Reed (22), the half-time of the mitochondrial GSH pool is much longer than the half-time of the cytoplasmic pool, 30 t 3 h vs. 2 t 0.1 h, in isolated rat hepatocytes, and the onset of cell injury by ethacrynic acid correlated with the depletion of mitochondrial GSH, whereas the cytosolic pool could be depleted without affecting cell viability. The BSO treatment presented in this study would affect little, if any, of the mitochondrial GSH pool prior to the onset of the antiproliferative effect, based on the length of BSO treatment and the fact that there is no effect of such BSO treatment on cell viability and CFE. It is thus suggested that the BSO antiproliferative effect does not follow depletion of a GSH pool necessary for maintaining cell viability. This study demonstrated also that NRK-49F cells are much more sensitive to BSO’s antiproliferative effects than are A549 cells. BSO at a concentration of 0.001 mM represents a level close to the threshold for growth in-
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BSO
AND
CELL
hibition in NRK-49F cells. The threshold level for A549 cells is, however, on the order of 1 mM, a concentration at which significant inhibition of NRK-49F cell proliferation is apparent. The delay times for cell cytostasis due to addition of 10 mM BSO are 36 h and 12 h in A549 and NRK-49F cells, respectively. BSO inhibits activation-dependent DNA synthesis in CD2 and CD3 antigen-stimulated T lymphocytes (28) in such fashion that dose-dependent inhibition of DNA synthesis is observed at BSO concentrations between 10B5 M and 10m3 M. However, treatment of these cells with 10m3 M BSO for 16 h was found to decrease cellular GSH content only from 0.70 t 0.26 to 0.63 t 0.14 nmol per lo6 cells. The effects of 10m4 M and 10m5 M BSO on cellular GSH levels were not reported. However, the data presented indicate a pronounced inhibition of mitogeninduced DNA synthesis in T lymphocytes treated with BSO in the absence of a significant reduction in GSH content. In this study, the effects of BSO on cellular GSH content and on cell proliferation were determined simultaneously. Analysis of such effects in both exponentially growing cells and EGF-stimulated growth-arrested cells strongly indicates that BSO-induced growth inhibition does not correlate with cellular GSH depletion.
GROWTH
11.
12.
13.
14.
15.
16.
17.
18.
19.
We thank Mary Nims for manuscript preparation. This work was supported by the National Institute of Environmental Health Sciences and by Iowa State University. Address for reprint requests: Y.-J. Kang, Dept. of Pharmacology, Univ. of North Dakota, School of Medicine, 501 N. Columbia Rd., Grand Forks, ND 58203.
20.
Received
22.
20 December
1990; accepted
in final
form
1 August
1991.
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