Vol. 168, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

April 30, 1990 Pages 551457

INDUCTION OF pGLUTAMYLCYSTEINE SYNTHETASE

BY PROSTAGLANDINA2 IN L-1210 CELLS

Kouji Ohno and Masaharu Hirata*

Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan

Received February 23, 1990

Summar : L-1210 ce Is were examined. When the cells were cultured in the T

Effects of prostaglandin A2 (PGA2) on glutathione (GSH) status in resence of PGA2,

a ersistent rise of cellular GSH concentration was observed 6 h a &?r the addition of P&AZ. This stimulatory effect of PGAz was abolished if the cells were pretreated with an enzyme inhibitor of GSH synthesis, buthionine sulfoximine. Subse uent study with cell free extract of cultured L-1210 has revealed that PGAz &mu P ated the biosynthesis of -glutam leysteine synthetase (EC 6.3.2.2). Actinomycin D inhibited this stimu atory e ect of PGAz on cultured cells. The optimal pH, Km I d value for glutamic acid and sensitivity to inhibitors of r- lutamylcysteine thetase from PGA2 treated and nontreated cells were virtual y the same. Thus, our 7

syn-

findings suggest that PGAz induced r-glutam 1210 cells which is responsible for the elevated r

lcysteine synthetase in cultured L eve1 of GSH in these cells. 0 1990

Academic Press, Inc.

PGAz, one of the cyclopentenone prostaglandins (PGs) and an enzymatic dehydration metabolite of PGE2, has been shown to inhibit the growth of cultured cells and induces cell differentiation in some cell lines (1-4). Unlike other PGs, PGAz is actively transported into cells by a specific carrier on the cell membrane and accumulates in cell nuclei with binding to nuclear proteins (5-7). After the accumulation to nuclei, PGAz induces synthesis of some 68kDa heat shock proteins at the transcriptional level and causes Gl-block of cell cycle progression (8,9). These findings suggest that PGA2 could have produced profound perturbation of the intracellular micro-environment. However, primary events responsible for such perturbation have not been elucidated. The a&unsaturated ketone moiety in PGAz is reactive as an electrophile, therefore, its interaction with cellular sulfhydryl com- ponents (e.g. GSH) drew much attention (10). Recently, several laboratory investigators have reported that reduction of cellular GSH contents by GSH depletors induced various changes of cell architectures. Mirabelli et al. (ll), for example, found that depletion of GSH by oxidative stress led to loss of protein sulfhydryl groups in cultured mammarian cells which resulted in successive changes of cytoskeleton and cell membrane integrity. We, therefore, examined the

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Vol. 168, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

possible correlation between GSH status and perturbation of intracellular environ- ment caused by PGAz.

Our results show that PGA2 induced marked elevation of cellular GSH content during culture and this elevation was due to induction of r-glutamylcysteine synthetase, a key enzyme of GSH biosynthesis.

MATERIALS AND METHODS

Materials - L-[G-3HlGlutamic acid (46 Ci/mmol) and L-fl4C(U)]glycine (108 mCi/mmol) were obtained from Amersham International plc. PGA2 was from Funakoshi Yakuhin Co. (Tokyo, Japan). Dried powder of RPM&1640 medium and Dulbecco’s PBS were from Nissui Seiyaku Ltd. (Tokyo, Japan). Fetal calf serum (FCS) was from M. A. Bioproducts (Walkersville, Md). GSH, ATP and actinomycin D were from Sigma Chemical Co. (St. Louis, MO). Glutamic acid, glycine, cysteine and glutamylcysteine were from nacalai tesque (Kyoto, Japan). All other chemicals used were of reagent grade.

Cell Culture and PG-Treatment - L-1210 murine leukemia cells were cultured in RPMI-1640 medium containing 10% FCS in humidified air with 5% CO2 at 37°C. Using a portion of the cell sus measuring the number of viable cells wit Yl

ension, growth of cells was monitored by trypan blue dye exclusion method. Cells

in the early stationary phase of growth were collected and suspended at a density of 4 x 105 cells/ml in culture flasks containing 4 ml of culture medium. The cells were incubated with 0, 3, 10 and 30 pM PGAz at 37°C. After 0, 6! 12 and 24 h of incubation, the number of viable cells was measured using a portion of cell suspen- sion and the cells were washed twice with ice-cold PBS, then suspended in 50 pl of PBS to determine the cellular GSH content. Duration of effect of PG-treatment was examined as follows: a suspension of L-1210 cells was incubated with 3OpM PGA2 for 6 h at 37°C. then washed twice with PBS. Cellular GSH was determined after additional 18 h incubation of the washed cells without PGA2. To examine the effect of BSO (buthionine sulfoximine) on cellular GSH, L-1210 cells were treated with 1 mM BSO for 12 h at 37°C and then incubated with 30 pM PGAz for 12 h at 37°C. For the assay of enzyme activities related to GSH biosynthesis in cell free extract, L- 1210 cells (3 x 106 cells/ml) were incubated for 12 h in 20 ml of culture medium with or without 30 pM PGA2. Following incubation, the cells were washed twice with ice- cold PBS and were pelleted by centrifugation at 100 x g for 5 min. Cell pellets were lysed by brief sonication in 1 ml of 100 mM TrisCl buffer (pH 7.4) containing 50 mM KCl, 20 mM MgCl2 and 2 mM EDTA. The lysates were then centrifuged at 15,000 x g for 20 min at 4”C, and 15,000 x g supernatants were used as the enzyme source for the determination of y-glutamylcysteine synthetase and GSH synthetase activities.

Actinomycin D-Treatment - A varying concentration of actinomycin D with or without 30 pM PGA:! was added to the suspension of L-1210 cells (3 x lOa/ml) and incubated for 12 h at 37°C. After incubation, the cells were disrupted and centrifuged for 20 min at 15,000 x g. The supernatant obtained was used for the determination of r-glutamylcysteine synthetase.

Determination of Cellular GSH Content - formed as described by Hissin and Hilf (12).

Determination of GSH was per- Briefly, to 50 1 of the cell sus

50 pl of 25% metaphos boric mh!r ensions,

cl EDTA buffer (

dp H 8.0) were a x ded.

acid and 400 pl of 100 NazHP04-5 m The mixture was cooled on ice for 10 min and cen-

trifuge at 10,000 x g for 10 min to precipitate the cellular proteins, then a 100 pl ahquot of the supernatant was transferred to a test tube containing 1.8 ml of 100 mM NazHP04-5 mM EDTA buffer (pH 8.0) and 100 pl of 7.5 n&I o-phfialald&y& at 25°C. Fifteen minutes after the reaction, fluorescence at 420 nm was measured with excitation at 350 nm. To confirmed the results obtained by Huorometric method, 50 pl of the cell suspension was mixed with 500 pl of 10% trichloroacetic

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Vol. 168, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acid, centrifuged for 10 min at 10,000 x g, then the supernatant was subjected to HPLC analysis as described below.

Determination of r-Glutamylcysteine Synthetase and GSH Synthetase Actiuities - r-Glutamylcysteine synthetase activity was determined by measur- ing the formation of [3Hlglutamate-labeled r-glutamylcysteine; the reaction mixture (final volume, 50 pl) containing 100 mM TrisCl buffer (pH 8.0 at 37°C ), 50 mM KCI, 20 mM MgC12, 2 mM EDTA, 10 mM ATP, 5 mM cysteine, 2.5 mM [aH]glutamic acid (2.4 mCi/mmol) and 15,000 x g supernatant (100 pg of soluble proteins), was incubated for 5,10,15 and 20 min at 37°C. After the incubation, the reaction was terminated by adding 50 pl of acetic acid and 800 pl of acetone and child on ice for 30 min, then centrifuged for 10 min at 15,000 x g to separate the labeled products from the cellular proteins. The supernatant was evaporated to dryness under a Nz gas stream, and the residual labeled products were dissolved in 50 pl of 50 mM KHZ PO4 (pH 3.5) to be analyzed b HPLC. To examine GSH synthetase activity, 5 mM 114Clglycine (0.8 mCi/mmo r ), 5 mM r-glutamylcysteine and 10 mM ATP were incubated with 15,000 x g supernatant (100 pg protein) in 50 pl of 100 mM TrisCl (pH 8.0)-50 mM KCl-20 mM MgClz-2 mM EDTA for 5, 10, 15 and 20 min at 37°C. The of samples for HPLC

rocedures for termination of the reaction and preparation ana P

glutamylcysteine. ysis were the same as those described for radio-labeled r-

Determination of GSH and r-Glutamylcysteine by HPLC - HPLC was performed on a Nucleosil 5NH2 column (4.6 x 250 mm) with 50 mM KH2P04- acetonitrile (3:l) acidified by phosphoric acid to pH 3.5 as a mobile phase at a flow rate of 1.0 mllmin. r-Glutamylcysteine and GSH were detected at 230 nm using a Jasco TWINCLE liquid chromatograph equipped with a variable-wave length UV detector (Japan Spectroscopic Co.). The retention times for r-glutamylcysteme and GSH were 8.5 and 7.4 min, respectively. Fractions corresponding to r-glutamyl- cysteine and GSH were collected and the radioactivity was measured.

Protein concentration was determined by the method of Lowry et al. (13) with bovine serum albumin as standard.

RESULTS

Elevation of Cellular GSH level by PG-Treatment - The effects of PGA2 on cellular GSH were examined by adding various concentration of PGA2 to L-1210 cells and incubating for 6,12 and 24 h. In control cells, the cellular GSH content at 6, 12 and 24 h was 2.1, 2.3 and 2.0 nmol/lOa cells, respectively. When the GSH content in cells exposed to the PG was compared with that in control cells, no significant difference was observed during the first 6 h incubation, while GSH level in cells exposed to the PGA2 was time- and PG concentration-dependently elevated during following incubation period (Fig. 1). The amount of GSH increased nearly 5- fold when cells were exposed to 30 pM PGA2 for 24 h. Treatment with 30 pM PGA2 did not show any cytotoxic effect on cell viability.

We therefore examined whether continuous exposure of cells to PGA2 would be necessary to support a high level of GSH. The cells incubated with 30 pM PGA2 for 6 h were washed and incubated in a fresh medium without PGA2 for further 18 h. As shown in Fig. 2, the GSH content began to rise at 9 h in the presence of 30 PM PGA2 and continuously increased during following incubation period. The amount of PGA2 taken up by the cells time-dependently increased during the PG-treatment, and its amounts in cytosol fraction at 3 and 6 h were 0.3 and 0.5 nmoVlO6 cells,

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6 12 24

Incubation time (hour)

Flc. Effect of PGA&eatment on cellular GSH in 11210 cells. L1210 cells (4 x 106 celldml) suspended in 4 ml culture medium were i&ted with 0.3.10 and 30 pM PCA2 for 6, 12 and 24 h at 37T. After the incubation, the number of viable cells was counted using a portion of the cell suspension. The cells were then washed twice with ice-cold PBS and rwuspsndod in 50 pl of PBS. The cell suspensions obtained were used for the determination of GSH withii the cells as described under “Materials and Methods”. (I),( ), (m), and (m) indicate the content of G6H in 61210 cells treated with 0.3.10 and 30 p?d PGAz. respectively. Values represent means f S.E. of three eqeri- me&.

respectively, though approx. 80% of PGA2 incorporated into the cells were found to

be effluxed 1 h after cell washing.

When cells were preincubated for 12 h with 1 mM BSO, an inhibitor of GSH

biosynthesis (14), and then exposed to 30 pM PGAz for 12 h in the presence of BSO,

no elevation of the cellular GSH level was observed. The finding suggests that tbe

PGA2 induced rise of GSH level is not due to suppression of GSH catabolism, but due

rather to stimulation of GSH biosynthesis.

Effect of PGA2 on GSH Biosynthesis - The study was extended to a cell

free system to examine the effect of PGAz on the enzyme activities related to GSH

biosynthesis. Cell extracts (150,000 x g supernatauts) were prepared from control

and PGAZ-treated cells aud used to measure the activities of r-glutamylcysteine

synthetase and GSH synthetase. When the cell extracts were incubated with

[3H]glutamic acid and cysteine in the presence of ATP, the formation of radio-

labeled r-glutamylcysteine in the extract from PG-treated cells was about 3-times

higher than that in the extract prepared from control cells (Fig. 3A). On the other

hand, when the extracts were incubated with [Wlglycine, r-glutamylcysteine and

ATP, no significant difference in the amount of radio- labeled GSH was observed

between that of control and PGAz-treated cells (Fig. 3B).

Since an elevated cellular GSH level was found to be due to an elevated level of

glutamylcysteine synthetase activity, we examined if PGAz directly stimulates the

activity of r-glutamylcysteine syutbetase in cell free extract. The extracts prepared

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Vol. 168, No. 2, 1990 BiOCHEMiCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cell washing

I 6 12 IS 24

Incubation time (hour)

0 0

0 3

20 0 10 20

Incubation time (mid

FIG. 2. Effect of cell washing on elevation of GBH level by PGAs in L1210 cells. 11210 cells (4 x lOa celldml) were incubated with or without 30 pM PGA2 in 4 ml ofculture medium for 6 or 24 h. The cells exposed to PoAp for 6 h were washed and incutu~ted in a fresh medium without the PG for another 18 h. The cells were collected et set times. GBH content in control cells (0 ) end G8H content in cells treated with the PG for 6 f a) and 24 h (0 ), reqmetively. Values represent means of two experiments.

a El&t of PGAp on y-glutamylcyetaine eynthetase activity (A) end GSH eynthetase activity (B). L1210 cells (3 x 106 cellsfml) suspended in 20 ml culture medium were incubated with or without 30 pM PGAs for 12 h at 37YI. ABer the incubation, the cells were washed and lysed by brief sonication. The cell lysates were centrifuged et 16,080 x g for 20 min at 4°C to prepare cell extracts and the eupernatanhr acre used as ensyme source for the determination of r-glutamyl cysteine syntbetase end GSH eynthetase activities as described under ‘Materiels end Methods”. Products formed in the supernatant prepared fmm contml cells (0 ) end cells treated with the PG f 0 ).

from control and PG-treated cells were incubated with [3Hlglutamic acid, cyst&e

and ATP in the presence of 30 to 300 pM PGA2, but I-glutamylcysteine synthetase

activity was not enhanced by PGAx (data not shown). Thus our results indicate

PGA2 could enhance the de IU)UO protein synthesis of r-glutamyl cysteine syn-

the&e. We therefore examined whether PGAx acts stimulatory on the transcrip- tion of genetic code. L1210 cells were incubated with 30 pM PGA2 in the presence of

actinomycin D for 12 h and r-glutamylcysteine synthetase activity was measured

using cell extracts. r-Glutamyl cyst&n synthetase activity was elevated from 0.75

to 2.15 nmole/min/mg protein in cells treated with PGA2. Actinomycin (100 nM)

completely inhibited this elevation of the enzyme activity.

Properties of r-Glutamylcysteine Synthetase in Cell-Free Extracts - The

enzymic properties of r-glutamylcysteine synthetase in extracts prepared from

control and PGA;r-treated cells were examined under our assay conditions. With

both extracts, [sH]glutamylcysteine linearly increased during incubation for 20 min,

and its amount was proportional to 50 lo 200 pg protein. The pH optimum for the

enzyme activity was about 8.3 for both extracts. With 0.2 to 5 mM concentration of

glutamic acid, the saturation kinetics of product formation was observed for the

extracts obtained from both control and PG-treated cells. The apparent Km value

for glutamic acid was about 1.8 mM with both enzymes, though the Vmax values of

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Vol. 168, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the enzyme activities in the extracts prepared from PGAz-treated and -nontreated

cells were 4.8 and 2.5 nmol/min/mg protein, respectively.

We next compared the inhibition profiles of GSH and BSO (14, 15) on the r-

glutamylcysteine synthetase activity between control cells and PG-treated cells,

which revealed that GSH and BSO inhibited the enzymes obtained from both sources

in a similar concentration-dependent manner.

DISCUSSION

The present study demonstrated that PGAz caused marked and sustained

elevation of GSH content in cultured mammalian cells, as a result of induction of r-

glutamylcysteine synthetase. The rise of GSH level was not observed when the cells

were pretreated with BSO, an inhibitor of r-glutamylcysteine synthetase. With

regard to the enzyme properties, the apparent Km value for glutamic acid and

sensitivity to enzyme inhibitors for the synthetase from PGAz-treated and -non-

treated cells were essentially the same. Furthermore, PGAz was found to increase

the GSH content of HeLa S3 and NIW3T3 fibroblasts (Ohno and Hirata unpublished

observations). It is, therefore, probable that PGAz stimulates GSH biosynthesis in

wide variety of mammalian cells. In our preliminary experiments, other primary

PGs such as PGBz, Dz, E2 and Fzo did not elevate the cellular GSH levels.

Induction of r-glutamylcysteine synthetase by PGA2 appears to occur at the

transcriptional level, as the stimulatory effect of PGAx was diminished by actino-

mycin D-treatment. Since PGAz accumulates in nuclei and binds to nuclear

proteins (7), the binding may evoke the transcription of gene coding r-glutamyl-

cysteine synthetase. It is generally accepted that sequence-specific DNA-protein

interaction occurs in enhancer/promoter regions of genes and this interaction

modulates the transcription rate (16,17). We speculate on the possible roles of PGA2

in gene transcription; one is that PGA2 binds and directly activates a specific

protein which is involved in the process of transcription, and another is that

stimulation of some enzyme (e.g. kinase) in nuclei which facilitates the process as

above described.

r-Glutamylcysteine synthetase catalyzes the first step of GSH biosynthesis.

According to clinical investigations, marked reduction of r-glutamylcysteine

synthetase activity in erythrocytes has been noticed in GSH deficient patients

associated with hemolytic anemia (18, 19). GSH plays multiple important roles in

supporting the homeostasis of cell functions (20), defense against toxic substances

and regulation of enzyme activities. Mitchel et al., for example, showed that cellular

GSH acted protectively against acetoaminophen induced hepatic necrosis (21).

When cells are exposed to oxidative stress, GSH depletion takes place which

initiates the oxidation of sulhydryl groups in actin leading to cell surface

abnormalities (11). Others have reported that deprivation of GSH suppressed the

signal transduction responded to extracellular stimuli, or Caz+ efflux from rat

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hepatocytes (22,23). PGH-D and PGH-E isomerase required GSH for their activities (24, 25). Recently, Yoshida et al. (26, 27) reported that GSH depletor induced hepatic heme oxygenase and ornithine decarboxylase at the transcriptional stage, proposing a hypothesis that variation of the cellular GSH level could serve as a trigger to initiate expression/suppression of specific genes. Taking these observa- tions into account, we assume PGA2, as its physiological role, modulates cell

functions via activation of GSH biosynthesis.

ACKNOWLEDGMENT

The authors thank Dr. S. Narumiya of Kyoto University Faculty of Medicine for reading the paper and for valuable suggestions.

1.

i, 4:

5:

7.

8.

9.

10. 11.

12. 13.

14.

:t 17: 18.

19.

20. 21.

22. 23.

24. 25. 26.

27.

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