Intracellular glutathione level modulates the induction of apoptosis by Δ12-prostaglandin J2

ELSEVIER

Intracellular Glutathione Level Modulates the Induction of Apoptosis by A 12-Prostaglandin J2 Ho-Shik Kim, Jeong-Hwa Department of Biochemistry, 505 Banpo-Dong, Socho-Ku,

Lee and In-Kyung

Kim

Catholic University Medical Seoul 137-701, Korea

College,

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an a,P-unsaturated carbonyl of 9-deoxy-A9*‘a-13, 14dihydroPGD, (A 12-PGJd, on the cytotoxicity of A’“-PGJ,. Az2-PGJ, caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The Al”-PGJ2 -induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of y-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by A’“-PGJ2. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of A’“-PGJ, is due to thiol-reactivity and intracellular GSH modulates the A’“-PGJ,-induced apoptosis by regulating the accessibility of A12-PGJ, to target proteins containing thiol groups.

Keywords: glutathione; AI’-PGJ,; apoptosis; thiol-reactivity; Hep 3B cells

Introduction prostaglandin(PG)s such as Cyclopentenone product 13,14-dihydroPGD, (A l2-PGJ,), a dehydration PGA,, a dehydration product of PGE,, were reported growth of various tumor cells.re4 We have recently

9-deoxy-A’!“of PGD,, and to inhibit the domonstrated

Address correspondence to: In-Kyung Kim, M.D. Department of Biochemistry, Catholic University Medical College, 505 Banpo-Dong, Socho-Ku, Seoul 137-701, Korea FAX 82-2-596-4435 Prostaglandins51:413-425,1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 100 10

0090-6980/96/$15.00 PI1 SOO90-6980(96)00047-O

Glutathione and Apoptosis ~YA~~-PGI,:Kim et al. that these PCs exert their cytotoxic effects via endogenous apoptotic pathway in L1210 murine leukemia cells5 But the exact mechanism by which these PCs initiate the cellular events resulting in cell death is not fully understood. It has been reported that nuclear accumulation of these PCs with binding to nuclear proteins relates closely to the growth inhibitory activity, suggesting that the transport of these PGs into nuclei is the essential step in eliciting cytotoxic effects.‘-* On the other hand, one of the characteristics of these cytotoxic PGs is that they have ~x,P-unsaturated carbonyl moieties in cyclopentenone rings, which are known to be susceptible to nucleophilic addition reaction with thiols.“,” Therefore, intracellular molecules containing thiol groups such as reduced glutathione (GSH), the major non-protein thiol, was supposed to be a candidate for the regulator of biological activities or the carrier molecule of these PGs. “,I2 Several groups have shown that these PGs readily conjugate with GSH in vitro and in vivo.13,14 However, the consequences of conjugate formation with GSH on the cytotoxic effects of these PGs were not consistent in various tumor cell types. Furthermore, the interaction between GSH and A 12-PGJzand its influence on cell survival are not yet determined in human tumor cells. In the present study, we changed the levels of intracellular GSH, prior to the addition of A 12-PGJ, and then examined cell survival and DNA integrity in human hepatocellular carcinoma Hep 3B cells.

Materials and Methods Chemicals A I2-PGJ, was generously donated by Ono Pharmaceutical Company (Osaka, Japan). Buthionine sulfoximine (BSO) and N-acetyl-cysteine (NAC) were purchased from Sigma (St.Louis, MO) and the DNA size markers (123 bp ladder DNA) were obtained from Gibco BRL (Gaithersburg, MD). All other reagents required for determination of intracellular GSH and extraction of DNA were of analytical grade and from Sigma, otherwise specified.

Measurement of Intracellular GSH Total GSH of Hep 3B cells was measured by the method of DTNB-GSSG reductase recycling assay.i5 Cells were seeded and treated with various concentrations of BSO, NAC, A”-PGJ, or sodium arsenite for indicated times. After treatments, cells (2.5~10~) were lysed and deproteinized with 5sulfosalicylic acid (final concentration, 5%), and then intracellular total

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GSH was assayed. The GSH contents were calculated according to the standard curve prepared using reduced GSH.

Cell Culture and Assay of Antiproliferative Activity of A”-PG/, Hep 3B cells were maintained in RPM1 1640 medium containing 10% heat-inactivated fetal bovine serum (Hyclone, UT) in humidified air with 5% CO,. To examine the effect of A”-PGJ, on the growth of Hep 3B cells, cells were plated at a density of 1~10~ cells/100 ~1 in 96-well culture plates and incubated with medium alone, BSO for 8 h or NAC for 4 h. And then, the medium was removed and cells were washed three times with phosphate-buffered saline (PBS) and fresh medium was added with or without various concentrations of A l2-PGJ, in triplicate. BSO was added again to the cells pretreated with BSO present to maintain the depleted GSH levels. To exclude the possibility that cysteine could block the transport of it by conjugating with A “-PGJ,, NAC was not added in the presence of A”-PGJ,. After 48 h, cell viability was determined by 3(4,5-dimethylthiazol-2-y1)2,5-diphenyl-tetrazolium bromide (MTT) test.‘” Data were represented as relative cell survival which means the percent of cell viability of treated cells to untreated cells.

Effect of Sodium Arsenite on the Growth of Hep 3B Cells To examine the antiproliferative activity of sodium arsenite, indicated concentrations of sodium arsenite, instead of A12-PGJ,, were added to Hep 3B cells pretreated with BSO or NAC as described above.

Analysis of DNA Fragmentation To examine DNA integrity of Hep 3B cells, total cellular DNA was extracted from 7.5 x 10’ cells in 6 ml incubated with various concentrations of A12-PGJ2 or sodium arsenite for 48 h as described.” Cells were lysed in lysis buffer (500 mM Tris-Cl, pH 9.0, 2 mM EDTA, 10 mM NaCl) containing 1% (W/V) sodium dodecyl sulfate (SDS) and treated with proteinase K (100 &ml) for 24 h. DNA was extracted with phenol/chloroform/isoamyl alcoho1(25:24: 1, V/V) and absolute ethanol. Equal amounts of extracted DNA were electrophoresed in 1.5% agarose gel. For the quantitative analysis of DNA fragmentation, the amounts of intact and fragmented DNA were quantified by the diphenylamine method modified before.18 The degree of DNA fragmentation was calculated as the percent of fragmented DNA to total DNA (sum of fragmented DNA and intact DNA). In some experiments, cells were treated with BSO or NAC before the addition of A12-PGJ,, as described above.

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Glutathione and Apoptosis byA”-PGJ,: Kim et al. To examine whether new protein syntheses are involved in DNA fragmentation induced by A1’2-PGJz or sodium arsenite, cells were incubated with cycloheximide (CHX, 0.5 pg/ml) for 30 min and washed with PBS before the addition of A12-PGJ, or sodium arsenite. In the experiments to determine the effect of CHX on the influence of BSO, CHX was treated to cells being incubated in the presence of BSO for the last 30 min. Results As shown in Table 1, The intracellular GSH levels were changed by pretreatment with BSO, an inhibitor of y-glutamylcysteine synthetase, or NAC, a precursor of cysteine. BSO rapidly reduced GSH contents up to 8 h dose-dependently. The GSH levels were elevated and reached maximal level at 4 h by NAC. So, to deplete GSH in Hep 3B cells, cells were preincubated in the presence of BSO for 8 h. And enrichment of intracellular GSH was carried out by incubating cells with NAC for 4 h.

TABLE I. Measurements

GSH contents 4h

Oh

Treatment Control

7.72 f

of GSH Contents in Hep 38 cells

0.07

(nmol/

2.5x1@ cells) 8h

_____ 12 h

7.94 f

0.34

1

6.87 f

0.25

6.56 f

0.47

6.70 f

0.04

10

4.54 f

0.04

3.60 f

0.02

3.44 f

0.17

4.12 + 0.33

1.80 f

0.01

1.95 f

0.06 0.52

7.81 + 0.09

_

BSO (PM)

100 NAC (mM) 1

8.00 + 0.02

8.04 f

0.03

7.66 f

2

8.75 + 0.05

8.50 + 0.20

8.63 f

0.07

5

9.87 f

0.03

9.79 + 0.14

9.90 f

0.01

4

7.93 f

0.28

8.01 + 0.35

7.80 + 0.05

8

7.72 + 0.13

7.85 f

7.50 + 0.02

A’%‘GJz

(&nl)

zsenite

0.60

_

(flM) 50

8.00 t 0.20

7.90 f

0.23

7.49 + 0.11

100

7.75 t 0.01

7.84 f

0.02

7.72 f ___.~ 0.03

Cells were plated and incubated in the presence of each concentrations of BSO, NAC, A’*PGJ, or sodium arsenite. At the indicated times, cells were lysed and deproteinized with 5sulfosalicylic acid (final concentration, 5%). And then total GSH was measured by DTNBGSSG reductase recycling assay”. The data were expressed as mean f S.D. of triplicate experiments.

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A”-PGJ, and sodium arsenite had no effects on the intracellular GSH contents. In the preliminary study, A12-PGJ, inhibited the growth of Hep 3B cells in a dose-dependent manner (data not shown). Figure 1 shows that the growth inhibitory effect of A12-PGJ, was potentiated by depletion of GSH following treatment with BSO. In contrast, relative cell survival was gradually recovered to control level by increasing concentrations of NAC up to 5 mM. Cell viability was not influenced by BSO or NAC treatment by itself (data not shown). We then examined DNA integrity of Hep 3B cells by agarose gel electrophoresis (Figure 2). When the cells were treated with 8 &ml of Ai2-PGJ2 for 48 h, cellular DNA was cleaved into multiples of 180 bp. The formation of ladder DNA was blocked by CHX, a protein synthesis inhibitor, which is the typical biochemical finding of apoptosis compared with necrosis.” While DNA of the cells treated with 4 k@rnl of A12-PGJ, alone remained to be intact, DNA of the cells treated with 4 pg/ml of AL2-PGJ, in the presence of BSO was found to be fragmented. BSO augmented DNA fragmentation induced by 8 &ml of A”-PGJ,, too. A

+ BSO

+ NAC

I

B

_

010 1 PM &?jj 1 mM 10 PM m 2 mM 100~M~5mM

8

a

Concentrations of A12-PGJ, @g/ml) FIGURE1. The effect of BSO (A) and NAC (B) on the antiproliferative activity of A’*-PGJ,. Hep 38 cells were plated and pretreated with medium alone, BSO for 8 h, or NAC for 4 h. The cells were washed with PBS and then treated with A”-PGJ2. BSO was added again with A’2-PGJzto BSOpretreated cells and consistently present. NAC was not readded. 48 h later, cell viability was measured by MlT test. 1,lO and 100 uM of BSO reduced GSH contents to 85,47 and 23% of that of control cells, respectiiely. On the contrary, NAC enriched GSH contents to 194,114 and 129% of that of control cells dose-dependently. The antiproliferative activity of A”-PGJ, was potentiated by GSH depletion and attenuated by GSH enrichment. The data were expressed as mean f S.D. of three triplicate experiments.

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CHX 0.5 pg/ml

BSO 100 pM NAC 5 mM A’*-pGJ,(pg/ml)

+

0

4

+

+

4

8

++ 8

8

4

8

300200loo-

FIGURE 2. Electrophoretic pattern of DNA extracted from Hep 36 cells. 7.5~10~ cells were treated with medium alone, CHX (0.5 pg/ml) for 30 min, BSO (100 PM) for 8 h, or NAC (5 mM) for 4 h. After being washed, cells were incubated in the absence or presence of A”-PGJ2 for 48 h. DNA was extracted and electrophoresed. While CHX and NAC were not added again, BSO was readded and consistently present with A’*-PGJ,. AC2-PGJ, induced DNA fragmentation which was

blocked by CHX. BSO depleted intracellular GSH and augmented DNA fragmentation. In contrast, NAC which elevated GSH level prevented DNA fragmentation. First lane shows a 100 bp DNA ladder as a marker.

NAC completely prevented the formation of DNA ladder. Quantitative analysis of DNA fragmentation using diphenylamine reagent showed again that BSO increased, and NAC reduced the fractions of fragmented DNA induced by A”-PGJ, (Figure 3). Figure 4 shows that the DNA fragmentation induced by A”-PGJ, was blocked by CHX also in the presence of BSO. This suggests that intracellular GSH plays a role in the upstream of de nova protein synthesis, such as transport and binding to target proteins of Al’-PGJz, in the apoptotic pathway activated by A I2-PGJ,. Next, we investigated the subsequent effect of GSH depletion or enrichment in murine leukemic L12 10 cells, where 2 &ml of A l2-PGJ, exhibited cytotoxic activity.5 As shown in figure 5, the formation of ladder DNA became more evident or diminished depending on doses of BSO or NAC respectively. We have observed this phenomenon in other

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and Apoptosis byA12-PGJ,: Kim et al.

tumor cells including human hepatocellular carcinoma Hep G2 and murine fibrosarcoma L929 cells (data not shown), indicating that the modulating effect of intracellular GSH level on the induction of apoptosis by AL2-PGJ, is not specific for Hep 3B cells. To examine whether the apoptosis-inducing activity of A 12-PGJ, is due to thiol-reactivity of an a,P-unsaturated carbonyl group, we tested the effect of sodium arsenite, another thiol reactive agent which can bind to sulfhydryls forming metal-thiol complex, on the viability of Hep 3B cells. When cells were incubated with sodium arsenite for 48 h, the viability was diminished dose dependently, up to 100 PM (Figure 6). In addition, the antiproliferative activity of sodium arsenite was enhanced or attenuated by depletion or enrichment of intracellular GSH respectively. As can be seen in figure 7, sodium arsenite caused DNA fragmentation which was also modulated by intracellular GSH, suggesting the

Concentrations of A ‘*-PGJ, @g/ml) FIGURE3. Quantification of DNA fragmentation induced by A’*-PGJ*. Hep 38 cells were plated and preincubated in the presence of BSO, NAC or CHX as described above. 48 h afier A’*-PGJ2 treatment, intact DNA and fragmented DNA were precipitated with trichloroacetic acid (final concentration, 12.5%) and extracted by heating. Extracted DNA was quantified using diphenylamine. The degree of DNA fragmentation means the percent of fragmented DNA to total DNA (sum of fragmented DNA and intact DNA). The data were expressed as mean f S.D. of three independent experiments.

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Glutathione and Apoptosis ~YA’~-PGJ,: Kim et al. BSO 100 pM CHX 0.5 pg/ml A’*-PGJz(pg/ml)

300200loo-

FIGURE4. The effect of CHX on the DNA fragmentation induced by A”-PGJ2 in the presence of BSO. Cells were plated and incubated in the presence of BSO (100 pM) for 8 h. And then cells were washed with PBS and treated with both BSO and A”-PGJ,. CHX was added 30 min. before washing and treatment with BSO and A’*-PGJ,. CHX was not readded after washing. CHX blocked DNA fragmentation which was induced by A’*-PGJ, and potentiated by BSO.

involvement of thiol-reactivity in the induction of apoptosis by A i2-PGJ, or sodium arsenite. Thus, it seems that the binding of A “-PGJz to thiol-containing molecules is the essential step in initiating apoptosis and that GSH, by conjugation with A12-PGJ,, may reduce the amount of available A12-PGJ, to the target molecules, thereby preventing the induction of apoptosis.

Discussion GSH is known to protect cells from damage by radiation and chemotherapy.20 In some tumor cells, the levels and capacity for synthesis of GSH are higher than normal tissues,21,22 which seems responsible for resistance to chemotherapy. Lowering the GSH levels augmented the effects of chemotherapeutic agents such as melphalan, bleomycin, and cyclophosphamide in various tumor cells in vitro and in viv~.~~~~” We observed here that the apoptosis-inducing capacity of A12-PGJ, or sodium arsenite in Hep 3B cells was potentiated by GSH depletion and

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Glutathione and Apoptosis byA’“-PG/i BSO NAC A’*-P(

Kim et al.

/ml)

FIGURE 5. Electrophoretic pattern of DNA extracted from Ll210 cells. Ll210 ceils were seeded at a density of 1~10~cells/3 ml and pretreated with medium alone, BSO (10, 100 pM) for 2 h, or NAC (2, 5 mM) for 1 h. While cells pretreated with BSO were washed and then treated with A”PGJ, and BSO together, other cells were washed and treated with A’*-PGJ2 alone. 48 h later, DNA was extracted. While 10 and 100 pM of BSO reduced to 70 and 38% of that of control cells respectively, 2 and 5 mM of NAC enriched GSH content to 112 and 134% of that of control cells respectively. The formation of ladder DNA became more evident or diminished depending on levels of intracellular GSH.

attenuated by GSH enrichment, resulted from BSO and NAC treatment, respectively. It did not seem that intracellular GSH affected the growth of Hep 3B cells by itself because cell viability was not changed by BSO or NAC treatment and neither A “-PGJz nor sodium arsenite changed the intracellular GSH levels (table 1). Moreover, several groups reported that GSH blocked the transport of A l2-PGJ, or sodium arsenite to nuclei and subsequent induction of several nuclear proteins.27,28 Taken together, it is likely that the apoptosis-inducing activity of AL2-PGJ, is due to thiolreactivity with nuclear proteins, of which activation is the critical requirement for initiation of cell death program. Considering that Ai2-PGJ, did not alter the intracellular GSH level as shown in our results and previous report”, A12-PGJ, may bind to only small fraction of intracellular GSH and the target proteins may have higher affinity to Al2 -PGJ, than GSH. Thus, it is possible that when the intracellular GSH level increases considerably, the fraction of

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421

Glutathione and Apoptosis ~YA’~-PGJ,:Kim et al. 0 + medium only m + BSO (100 PM) Epj + NAC (5 mM)

0

25

50

75

100

Concentrations of sodium arsenite (PM) FIGURE6. The effect of BSO and NAC on the sodium arsenite-induced growth inhibition. Hep 38 cells were plated and treated as described in Materials and Methods. Cell viability was measured by MTT assay, 48 h after treatment of sodium arsenite. Sodium arsenite, a thiol-reactive agent, exerted antiproliferative effect which was varied by intracellular GSH the same as A.“-PGJ?. The data were expressed as mean f S.D. of three triplicate experiments.

Al’-PGJ, that conjugate with GSH may increase, thereby reducing the accessibility of A”-PGJz to target nuclear proteins. Our results, however, are not consistent with the previous report of PGA,, in which GSH-PGA, conjugate or its metabolite is more potent than non-conjugate form, l4 indicating that the antiproliferative mechanism of A”-PGJ2 is somewhat different from that of PGA,. It has been reported that A l2-PGJ, conjugates with GSH and is metabolized to reduced form at the 1 I-keto and A l2 double bond in rat liver, Chinese hamster ovary and rat hepatoma cells.11~‘3 PGD, is also known to be secreted by activated mast cells under inflammatory circumstances2g,30 and to be converted to A l2-PGJ, in the presence of serum albumin.3”32 Considering that tumor development and progression is often accompanied by local inflammatory processes, it can be postulated that A “-PGJ, produced from PGD, may play a role in deleting tumor cells by inducing apoptosis, and lowering intracellular GSH levels may contribute to preventing tumor progression by rendering some tumor cells to be more susceptible to anticancer drugs which are reactive to thiols.

422

Prostaglandins 1996: 5 1, June

Glutathione and Apoptosis byh”-PGJ,: Kim et al. Cl-IX 0.5 Fg/ml BSO 100 pM NAC5mM

Arsenite(@

+ ++ ++

0 50 100100 50 100 50 100

bp

300-

200100-

FIGURE7. Formation of ladder DNA in Hep 38 cells induced by sodium arsenite. Cells were pretreated with medium alone, CHX (0.5 pglml), BSO (100 uM), or NAC (5 mM) as described in Materials and Methods. 48 h after the treatment with sodium arsenite, total DNA was extracted and electrophoresed. Sodium arsenite caused DNA fragmentation which was blocked by CHX. DNA fragmentation induced by sodium arsenite was also modulated by GSH contents in cells. CHX was also able to block DNA fragmentation induced by sodium arsenite in the presence of BSO.

Acknowledgements This study was supported in part by grants from the Basic Research Promotion Fund of the Ministry of Education, Korea, Cancer Research Center in Seoul National University (KOSEF-SRC-56-CRC-94K3-040204-02-3), and Mission-oriented grant from Korea Science and Engineering Foundation (F025534).

References 1.

2.

Nammiya, S. and Fukosbima, M. A”-Prostaglandin J2,an Ultimate Metabolite of Prostaglandin D, Exerting Cell Growth Inhibition. Biochem. Biophys. Res. Commun. 127:739. 1985. Bregman, M.D. and Meyskens, F.L.,Jr. In Vitro Modulation of Human and Murine Melanoma Growth by Prostanoid Analogues. Prostaglandins 26:449. 1983.

Prostaglandins 1996: 5 1, June

423

Glutathione

and Apoptosis byA “-PGJ,: Kim et al. 3.

4.

5.

6.

7.

8.

9.

IO.

11.

12.

13.

14. 15.

16.

17.

424

Bhuyan, B.K., Adams, E.G., Badiner, G. J., Li, L.H. and Barden, K. Cell Cycle Effects of Prostaglandins A,, AZ, and D, in Human and Murine Melanoma Cells in Culture. Cancer Res. 46:1688. 1986. Santoro, M.G., Crisari, A., Benedetto, A. and Amici, C. Modulation of the Growth of a Human Erythroleukemia Cell Line (K562) by Prostaglandins: Antiproliferative Action of Prostaglandin A. Cancer Res. 46:6073. 1986. Kim, I.K., Lee, J.H., Sohn, H.W., Kim, H.S. and Kim, S.H. Prostaglandin A, and A”-Prostaglandin J2 Induce Apoptosis in L1210 Cells. FEBS Lett. 321:209. 1993. Narumiya, S. and Fukushima, M. Site and Mechanism of Growth Inhibition by Prostaglandins.1. Active Transport and Intracellular Accumulation of Cyclopentenone Prostaglandins, a Reaction Leading to Growth Inhibition. J. Pharmacol. E!xp. Ther. 239:500. 1986. Narumiya, S., Ohno, K., Fukushima, M. and Fujiwara, M. Site and Mechanism of Growth Inhibition by Prostaglandins.11. Temperature-dependent Transfer of a Cyclopentenone Prostaglandin to Nuclei. J. Pharmacol. Exp. Ther. 239:506. 1986. Narumiya, S., Ohno, K., Fukushima, M. and Fujiwara, M. Site and Mechanism of Growth Inhibition by ProstaglandinsIII. Distribution and Binding of Prostaglandin A, and A”-Prostaglandin Jz in Nuclei. J. Pharmacol. Exp. Ther. 2421306. 1987. Cagen, L.M. and Pisano, J.J. The Glutathione Conjugate of Prostaglandin A, is a Better Substrate than Prostaglandin E for Partially Purified Avian Prostaglandin E Y-ketoreductase. Biochim. Biophys. Acta 573:547. 1979. Honn, K.V. and Mamett, L.J. Requirement of a Reactive cz,J3-Unsaturated Carbonyl for Inhibition of Tumor Growth and Induction of Differentiation by ‘A” Series Prostaglandins. Biochem. Biophys. Res. Commun. 129:34. 1985. Atsmon, J., Sweetman, B. J., Baertschi, S.W., Harris, T.M. and Roberts, L. J.,II. Formation of Thiol Conjugates of Y-Deoxy-A9,A”(E)-prostaglandin D, and A”(E)-Prostaglandin D,. Biochemistry 29:3760. 1990. Ohno, K. and Hirata, M. Characterization of the Transport System of Prostaglandin A, in L-l 210 Murine Leukemia Cells. Biochem. Pharmacol. 46:661, 1993. Atsmon, J., Freeman, M.L., Meredith, M. J., Sweetman, B. J. and Roberts, L.J.,II. Conjugation of 9-Deoxy-Ay,A12(E)-prostaglandin D, with Intracellular Glutathione and Enhancement of its Antiproliferative Activity by Glutathione Depletion. Cancer Res. 50: 1879. 1990. Parker, J, and Ankel, H. Formation of a Prostaglandin A,-glutathione Conjugate in L1210 Mouse Leukemia Cells. Biochem. Pharmacol. 43:1053. 1992. Anderson, M.E. In: Meti. Enzymol. Vol. 113. Determination of Glutathione and Glutathione Disulfide in Biological Samples. (A. Meister, ed) Academic Press, London. 1985, p. 548. Mosmann, T. Rapid Calorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxic Assays. J. Immunol. Methods 65:55. 1983. Kaufrnann, S.H. Induction of Endonucleolytic DNA Cleavage in Human Acute Myelogenous Leukemia Cells by Etoposide, Camptothecin, and Other Cytotoxic Anticancer Drugs: A Cautionary Note. Cancer Res. 49:5870. 1989.

Prostaglandins

1996: 5 1, June

Glutathione 18. 19.

20. 21.

22.

23.

24.

25.

26. 27.

28.

29. 30.

31. 32.

Sellins, K.S. and Cohen, J.J. Gene Induction by y-Irradiation Leads to DNA Fragmentation in Lymphocytes. J. Immunol. 139:3199. 1987. Lo&shin, R.A. and Zakeri, Z. In: Apoptosis: The Molecular Basis of Cell Death. Programmed Cell Death and Apoptosis. (L.D. Tomei and F.O. Cope, eds.) Cold Spring Harbor Laboratory Press, New York. 199 1, p. 47. Meister, A. Glutathione, Ascorbate, and Cellular Protection. Cancer Res. 54:1969s. 1994. Russo, A., DeGraff, W., Friedman, N. and Mitchell, J.B. Selective Modulation of Glutathione Levels in Human Normal versus Tumor Cells and Subsequent Differential Response to Chemotherapy Drugs. Cancer Res. 46:2845. 1986. Allalunis-Turner, M.J., Lee, F.Y.F. and Siemann, D.W. Comparison of Glutathione Levels in Rodent and Human Tumor Cells Grown in Vitro and in Vivo. Cancer Res. 48:3657. 1988. Green, J.A., Vistica, D.T., Young, R.C., Hamilton, T.C., Rogan, A.M. and Ozols,R.F. Potentiation of Melphalan Cytotoxicity in Human Ovarian Cancer Cell Lines by Glutathione Depletion. Cancer Res. 44:5427. 1984. Russo, A., Mitchell, J.B., McPherson, S. and Friedman, N. Alteration of Bleomycin Cytotoxicity by Glutathione Depletion or Elevation. Int. J. Radiat. Oncol. Biol. Phys. 10:1675. 1984. Lee, K.E., Mayer, R.D. and Cockett, A.T.K. Effect of Systemic Glutathione Depletion by Buthionine Sulfoximine on Sensitivity of Murine Bladder Cancer to Cytotoxic Agents. Invest. Urol. 34:376. 1989. Siemann, D.W. and Beyers, K.L. In Vivo Therapeutic Potential of Combination Thiol Depletion and Alkylating Chemotherapy. Br. J. Cancer 68:1071. 1993. Koizumi, T., Negishi, M. and Ichikawa, A. Inhibitory Effect of an Intracellular Glutathione on A”-Prostaglandin TX-induced Protein Synthesis in Porcine Aortic Endothelial Cells. Biochem. Pharmacol. 44: 1597. 1992. Koizumi, T., Negishi, M. and Ichikawa, A. Activation of Heat Shock Transcription Factors by A”-Prostaglandin Jz and its Inhibition by Intracellular Glutathione. Biochem. Pharmacol. 45:2457. 1993. Ito, S., Narumiya, S. and Hayaishi, 0. Prostaglandin D,: A Biochemical Perspective. Prostaglandins Leukot. Essent. Fatty Acids 37:219. 1989. Davies, P. and Maclntyre, D.E. In: Inflammation, Basic Principles and Clinical Correlates, 2nd Ed. Prostaglandins and Inflammation (J.I. Gallin, I.M. Goldstein and R. Snyderman, eds) Raven Press, New York. 1992, p. 123. Fitzpatrick, F.A. and Wynalda, M.A. Albumin-catalyzed Metabolism of Prostaglandin D,. J. Biol. Chem. 258:11713. 1983. Kikawa, Y., Narumiya, S., Fukushima, M., Wakatsuka, H. and Hayaishi, 0. 9Demy-A9A n-1 3,14-dihydroprostaglandin D,, a Metabolite of Prostaglandin D, Formed in Human Plasma. Proc. Natl. Acad. Sci. USA 8 1: 13 17. 1984.

Editor: B.M. Jaffe

Prostaglandins

and Apoptosis byA*‘-PGJ,: Kim et al.

1996: 5 1, June

Received: 07-10-95

Accepted: 04-08-96

425