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Volume 270, Number 33, Issue of August 18, pp. 19451-19457, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Markedly Decreased Expression of Glutathione S-Transferase Gene in Human Cancer Cell Lines Resistant to Buthionine Sulfoximine, an Inhibitor of Cellular Glutathione Synthesis (*)

(Received for publication, April 7, 1995)

Akira Yokomizo (1),  Kimitoshi Kohno (1),  Morimasa Wada (1),  Mayumi Ono (1),  Charles S. Morrow (2),  Kenneth H. Cowan (3),  Michihiko Kuwano (1)(§)

From the  (1)Department of Biochemistry, Kyushu University School of Medicine, Maidashi 3-1-1, Fukuoka 812-82, Japan, the (2)Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016, and the (3)Medicine Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, -glutamylcysteine synthetase (-GCS), which is a critical step in glutathione biosynthesis. We isolated three BSO-resistant sublines, KB/BSO1, KB/BSO2, and KB/BSO3, from human epidermoid cancer KB cells. These cell lines showed 10-to 13-fold higher resistance to BSO, respectively, and had collateral sensitivity to cisplatin, ethacrynic acid, and alkylating agents such as melphalan and nitrosourea. Cellular levels of glutathione S-transferase (GST-) and its mRNA in BSO-resistant cell lines were less than 10% of the parental cells. Nuclear run-on assay showed that the transcriptional activity of GST- was decreased in BSO-resistant cells, and transient transfection of GST- promoter-chloramphenicol acetyltransferase constructs revealed that the sequences between -130 and -80 base pairs of the 5`-flanking region were at least partially responsible for the decreased expression of the GST- gene. By contrast, -GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the -GCS gene was found to be amplified in the BSO-resistant cell lines. GST- mRNA levels appeared to be inversely correlated with -GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-1 and KB/BSO3-2, that overexpressed GST-, from KB/BSO3, after introducing a GST- expression plasmid. These two transfectants had similar levels in -GCS mRNA, drug sensitivity to alkylating agents, and glutathione content as those of KB cells. These findings suggest that the cellular levels of GST- and -GCS might be co-regulated in these novel BSO-resistant cells.

INTRODUCTION

Intracellular non-protein sulfhydryl glutathione (GSH) has multiple functions in catalysis, transport, and reductive phenomena(1, 2) . Moreover it reacts with toxic endogenous and exogenous substances, including free radicals and anticancer agents. These functions are important in drug resistance during cancer chemotherapy with agents such as nitrogen mustards, nitrosourea, cisplatin, and anthracyclines (3, 4, 5, 6, 7) . GSH is a tripeptide of glycine, glutamic acid, and cysteine, which is synthesized intracellulary by the action of two enzymes: -GCS (^1)and glutathione synthetase. -GCS is a rate-limiting enzyme in the synthesis of GSH and is feedback-inhibited by GSH(8, 9) .

Glutathione metabolism is often correlated with cellular sensitivity to anticancer agents. Indeed, glutathione is protective against drug cytotoxicity(1, 9, 10, 11) . Acquired resistance to alkylating agents is frequently accompanied by an elevation in the cellular non-protein sulfhydryl content. Melphalan-resistant leukemia cells have a 2- to 4-fold higher level of GSH than the sensitive parental cells(12, 13) . A relevant study by Ozols and his colleagues (3, 14, 15) has demonstrated that ovarian cancer cell lines resistant to adriamycin, cisplatin, and various alkylating agents such as nitrosourea and cyclophosphamide have increased GSH levels. Furthermore, lowering GSH levels by treatment with the -GCS inhibitor, BSO, can potentiate the activity of melphalan, cisplatin, and other anticancer agents in vitro as well as in vivo(6, 10, 16, 17, 18, 19) .

In our laboratory, we demonstrated that Chinese hamster ovary (CHO) cell lines resistant to cisplatin have increasing levels of GST-, suggesting the involvement of GST in the acquisition of the cisplatin-resistant phenotype(20) . The introduction of GST- cDNA makes the recipient CHO cells 1.4- to 3.0-fold more resistant to cisplatin(21) . The multidrug-resistant variant of MCF-7 breast cancer cells, which is resistant to adriamycin and other anticancer agents, overexpresses GSH-dependent enzymes GST and glutathione peroxidase(22) . The GST- cDNA-transfected MCF-7 cells are more resistant to benzo(a)pyrene and ethacrynic acid, but not to melphalan and cisplatin(23) . A study by Nakagawa et al.(24) has demonstrated that GST- cDNA transfectants of mouse NIH3T3 cells are more resistant to adriamycin and ethacrynic acid, but not to melphalan and cisplatin. These findings suggest that increased GST levels play a role in determining the sensitivity to some drugs, including alkylating agents, but that the selectivity of drugs appears to differ according to the origin of the cell line. Godwin et al.(25) have reported that high cisplatin resistance in human ovarian cancer cells is associated with the enhanced expression of mRNAs for -GCS, again suggesting the involvement of glutathione synthesis in the acquisition of drug resistance.

Glutathione function has been studied extensively in relation to drug metabolism(1, 9, 10) . However, it remains unknown how GSH specifically detoxifies anticancer agents and how cellular glutathione levels and GSH associated enzymes are selectively modulated in cancer cells. In this study, we first isolated human cancer cell lines resistant to the cytotoxic effects of BSO. GST- gene expression was greatly abrogated, but that of -GCS was up-regulated in these BSO-resistant cell lines. The coordinate and inverse co-regulation of GST- and -GCS genes is discussed in relation to the acquisition of the novel BSO-resistant phenotype.

EXPERIMENTAL PROCEDURES

Materials

BSO, adriamycin, vincristine, and ethacrynic acid were from Sigma. ACNU and etoposide were obtained from Sankyo Co., Osaka, Japan and Nippon Kayaku Co., Tokyo, Japan. Cisplatin and melphalan were donated by Bristol Myers Co., Kanagawa, Japan and Nihon Wellcome Co., Osaka, Japan. G418 was purchased from Life Technologies, Inc. [alpha-P]dCTP, [P]UTP, and [^14C]chloramphenicol were from DuPont NEN. Hybond N membranes and the DNA labeling kit were obtained from Amersham International plc. Lipofectin was purchased from Bethesda Research Laboratories. beta-NADP was purchased from ORIENTAL Yeast Co., Ltd, Japan, 5,5`-dithiobis(2-nitrobenzoic acid) was from Nacalai Tesque Inc. Kyoto, Japan, and glutathione reductase was from Boehringer Mannheim GmbH, Mannheim, Germany.

Cell Culture and Isolation of BSO-resistant Cell Lines

The human epidermoid cancer KB cell line and its BSO-resistant cell lines (KB/BSO1, -2, and -3) were cultured in Eagle's minimal essential medium (Nissui Seiyaku Co., Tokyo) containing 10% newborn calf serum (Sera-lab Ltd., Sussex, United Kingdom), 0.292 mg of glutamine/ml, 100 µg/ml kanamycin, and 100 units/ml penicillin at 37 °C in a humidified atmosphere of 5% CO(2)(26, 27, 28, 29) . To isolate BSO-resistant sublines, KB cells were first incubated with 230 µg/ml ethyl methanesulfonate for 20 h, then in the absence of any drug for 3 days. Exponentially growing KB cells were plated at a density of 10^6/100-mm dish, and then 500 µM BSO was added to the medium. At 3-week intervals, BSO concentrations were increased to 2 mM and then 5 mM BSO. During continuous exposure to BSO, culture medium was replaced with freshly prepared medium containing BSO at indicated concentrations every 4-5 days. Three BSO-resistant lines, KB/BSO1, -2, and -3 were independently purified and cloned from different dishes as described(26, 27) . The BSO-resistant phenotype did not change when these cell lines were cultured for 3 months in the absence of any drug (data not shown). These results indicate that the BSO-resistant phenotype is stable and is maintained for at least for 3 months in the absence of drug selection.

Colony Formation Assay

We seeded 400 KB cells and 700 cells of its variants in 35-mm dishes in the absence of drugs at 37 °C for 18 h. The cells were then continuously incubated for an additional 7 days with various drugs as described(28, 29) .

Immunoblot of GSTs

Cytosol protein fractions (100 µg/lane) extracted from indicated cells were analyzed by SDS-polyacrylamide gel electrophoresis (12% gels)(20) . Protein fractions from the gel were transferred onto nitrocellulose membranes in 25 mM Tris-HCl (pH 8.3), 92 mM glycine, 20% methanol for 4 h at 120 V. The nitrocellulose membranes were further incubated with antibodies against human alpha, µ, and class GSTs(30) (1:4000) for 1 h at room temperature(20, 21) . The membranes were rinsed with Tris-buffered saline, immersed in biotinylated secondary antibody, and then developed according to the manufacturer's specifications (Vectastain ABC-GO kit, Vector Laboratories)(20) .

RNA Isolation and Northern Blot

The human MGMT probe was constructed as described previously(31) . The GST- cDNA probe was kindly donated from M. Muramatsu (Saitama Medical University, Saitama, Japan)(32) , -glutamyl transpeptidase cDNA was from H. C. Pitot (McArdle Laboratory of Cancer Research)(33) , -GCS cDNA was from the American Type Culture Collection(34) , and glutathione peroxidase cDNA was from Nippon Kayaku Co., Ltd. (Tokyo, Japan) (35) . Total RNA was isolated using guanidine isothiocyanate(28, 29) . The concentrations of all RNA samples were determined spectrophotometrically at 260 nm. RNA samples (15 µg/lane) were separated on a 1% formaldehyde-agarose gel and transferred to a Hybond N membrane with 10 SSC. The membranes were baked and hybridized with alpha-P-labeled probes and visualized by autoradiography. Radioactivity was detected using a Fujix Bas 2000 bioimaging analyzer (Fuji Photo Film Co., Tokyo)(29) .

Nuclear Run-on Transcription Assay

The nuclei from each cell line were prepared as described previously(36) . For the transcription assay, the following were added to 100 µl of nuclei (1 10^8/ml) in the nuclei suspension buffer (36) and incubated at 30 °C for 30 min: 20 µl of 10 transcription buffer (0.7 M KCl, 50 mM MgCl(2), 50 mM Tris-HCl, pH 8.0, 25 mM dithiothreitol, 1 mM EDTA), 20 µl of 10 ATP, CTP, and GTP (2.5 mM each), 1 µl of RNasin (200 units/ml), 60 µl of H(2)O, and 200 µCi of [P]UTP (800 Ci/mmol). RNA labeled with P was isolated as described previously(36) .

Analysis of RNA Synthesized in Isolated Nuclei

Plasmid DNAs were linearized by appropriate restriction enzymes and purified(37) . Linearized DNA of 5 µg/slot was suspended in 0.2 N NaOH and 6 SSC, boiled for 10 min, and chilled on ice for 10 min, and an equal volume of 2 M Tris-HCl (pH 7.4) was added. The DNA was blotted onto a Hybond N membranes, then prehybridized in Hybrisol I. Hybridization was performed in Hybrisol I with P-labeled nuclear RNA (10 10^6 cpm/membrane) for 72 h at 42 °C. Membranes were washed in 0.2 SSC, 0.1% SDS at 50 °C, digested with RNase A (10 µg/ml) in 2 SSC, and then autoradiographed with Fujix Bas 2000 bioimaging analyzer.

CAT Assays

The GST- promoter-CAT plasmids were constructed as described previously(38) . The plasmid DNAs used in this study were p-2203 GST-CAT, p-130 GST-CAT, and p-80 GST-CAT, which contain -2203-, -130-, and -80-nucleotide 5`-flanking region of GST- gene. We also co-transfected pSV2-beta-gal as internal control. The transfection experiments were done by using Lipofectin. The transfected cells were washed twice with phosphate-buffered saline and harvested in 40 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 150 mM NaCl. The cell pellet was resuspended and sonicated in 0.25 M Tris-HCl (pH 7.9), then assayed for protein content by the method of Bradford(39) . Each CAT assay was performed with identical amounts of protein as described before(40) . beta-Galactosidase activity was measured as described(41) .

Southern Blot

Genomic DNA was isolated from the parental KB cell line and its resistant cell lines. Southern blotting was performed according to the standard protocols(29) . DNA was digested to completion with EcoRI. Twenty micrograms of DNA was loaded per lane in a 1% agarose gel, then the DNA was transferred onto a Hybond N membrane. The DNA blots were hybridized with alpha-P-labeled cDNA probes for 24 h at 42 °C, then washed at room temperature in 2 SSC and 0.1% SDS, followed by 0.2 SSC and 0.1% SDS. The DNA levels were quantified by radioactivity using a Fujix Bas 2000 bioimaging analyzer.

Transfection

For stable transfections, we used the GST- expression plasmid, pbeta actGpi-2. This vector was constructed by insertion of a GST- cDNA at the HindIII-BamHI site of the human beta-actin expression vector pHBAPr-1(21, 24) . The plasmid pSV2-neo has been described(42) . KB cells were transfected with a mixture of pbeta actGpi-2 (10 µg) plus pSV2-neo (0.5 µg) using Lipofectin. We also transfected only pSV2-neo (10 µg) into KB cells as control. After 12 h, the medium containing DNA and Lipofectin was replaced with fresh medium overnight. The cells were then incubated in selection medium containing 800 µg/ml geneticin (G418) (Life Technologies, Inc.) for 3 to 4 weeks. G418-resistant colonies were cloned in the presence of G418, and several transfectants that overexpressed the GST- gene were selected. These transfectants, BSO3-1 and BSO3-2 were continuously cultured in the presence of 800 µg/ml G418(21) .

GSH Assay

Total GSH was determined by means of the enzymatic recycling assay based on the glutathione reductase method of Griffith(43) . Briefly, 2 10^6 cells were plated in 100-mm dishes, and the next day BSO was added at each concentration. After 18 h, cells were collected and washed twice with phosphate-buffered saline at 4 °C. Cell pellets were resuspended in 900 µl of double-distilled water and shaken vigorously for 3 min. 450 µl of cell lysate was removed for GSH assay, and the protein concentration was evaluated from the remaining sample. For GSH assay, 100 µl of 30% 5-sulfosalicylic acid was added to each sample tube. The solution was mixed and incubated at 4 °C for 10 min, then centrifuged at 2,800 g for 10 min. The enzyme reaction proceeded in a 1-ml cuvette in 700 µl of 0.3 mM beta-NADP, 100 µl of 6 mM 5,5`-dithiobis(2-nitrobenzoic acid), 165 µl of stock buffer (125 mM sodium phosphate, 6.3 mM EDTA, pH 7.5), 190 µl of supernatant, and 10 µl of glutathione reductase (50 units/ml) at 37 °C. The absorbance at 412 nm (A) was monitored for 3 min at 20-s intervals, and GSH content was calculated from the rate of change in absorption at A compared with a standard curve as described(20, 44) .

RESULTS

Establishment of BSO-resistant Cell Lines from Human Cancer KB Cells and Their Drug Sensitivities

The sensitivities of the three BSO-resistant cell lines, KB/BSO1, KB/BSO2, and KB/BSO3, to various drugs was determined by colony formation assays. The IC values of the parent KB and its BSO-resistant sublines were determined and used to calculate the relative resistance to drugs as shown in Table 1. KB/BSO1, KB/BSO2, and KB/BSO3 were 10-, 11-, and 13-fold more resistant to BSO, respectively, than the parental KB cell line. All BSO-resistant cell lines also showed collateral sensitivity to ACNU, melphalan, and ethacrynic acid and also a slight collateral sensitivity to cisplatin. By contrast, the sensitivities to etoposide, adriamycin, and vincristine of the BSO-resistant cells were similar to those of the KB cells (Table 1).

Cellular Levels of -GCS, Glutathione Peroxidase, -Glutamyl Transpeptidase, GST-, and MGMT in KB and Its BSO-resistant Sublines

Since BSO is a specific inhibitor of GSH synthesis(18, 19) , we examined whether the expression of GSH-related enzymes was altered in BSO-resistant cell lines. Expression of four relevant enzyme genes, -GCS, glutathione peroxidase, -glutamyl transpeptidase, and GST-, was determined by Northern blot (Fig. 1). Cellular mRNA levels of glutathione peroxidase and -glutamyl transpeptidase in the three BSO-resistant cell lines were similar to those in the parental KB cells, whereas GST- mRNA levels in resistant cell lines were less than 10% of that in KB cells. The -GCS mRNA level was increased 3- to 5-fold in BSO-resistant cells compared with KB cells (Fig. 1). We also examined the mRNA level of MGMT, which is often associated with resistance to alkylating agents such as nitrosourea or N-methyl-N,N`-methyl-N-nitrosoguanidine (31, 45) . There appeared however no difference in the MGMT mRNA levels between KB and its BSO-resistant cell lines (Fig. 1). As shown in Fig. 1F, Western blot also revealed an apparent decrease in the amounts of GST- in BSO-resistant cell lines in comparison with KB cells. Within the GST enzyme family, the class GST, but not the alpha or µ class GSTs is specifically expressed in human cancer KB cells(44) .

Figure 1: Northern blot of -glutamylcysteine synthetase (-GCS) (A), glutathione peroxidase (GPX) (B), -glutamyl transpeptidase (-GTP) (C), MGMT (D) and GST- (E) mRNAs, and immunoblot of GST- (F). Total RNA (15 µg) extracted from KB, KB/BSO1, KB/BSO2, and KB/BSO3 cells were resolved by electrophoresis in a 1% agarose gel containing 2.2 M formaldehyde, transferred to Hybond N, and hybridized with various P-labeled cDNA probes (B, C, D, and E). The equivalent loading of total RNA is shown by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blot (A). For immunoblotting of GST-, 100 µg of cytosolic fractions from each cell line were resolved on 12% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and then incubated with rabbit antibody against human GST- and biotinylated goat anti-rabbit IgG (F).


Nuclear Run-on Assay and GST- Promoter Activity in KB/BSO3

We performed nuclear run-on assays to determine whether decreased expression of the GST- gene and enhanced expression of the -GCS gene were due to altered transcriptional activities of these genes. Transcription rates of GST- and -GCS in KB/BSO3 were 0.2- and 3.0-fold, respectively, in comparison with parent KB cells (Fig. 2). The transcription rates of glutathione peroxidase and GAPDH were not changed in these two cell lines (Fig. 2). To investigate the transcriptional regulation of GST- between KB and its BSO-resistant cells, we transfected GST- promoter and its deleted promoter CAT plasmids (38) into these cell lines. Transfection of p-2203 GST-CAT and p-130 GST-CAT resulted in significantly decreased (p < 0.05) CAT expression in KB/BSO3 relative to expression in KB cells (Fig. 3). On the other hand, p-80 GST-CAT transfection showed similar CAT activity in KB/BSO3 and KB cells. These results suggest that trans-regulating factor(s), which might recognize the sequences between -130 bp and -80 bp from the transcription initiation site, is at least partially responsible for the altered GST- expression in BSO-resistant cell lines.

Figure 2: Nuclear run-on analysis of GST- and-GCS. Transcription in the isolated nuclei was analyzed by hybridization of P-labeled transcript to 5 µg of PUC-19 plasmid, GST-, -GCS, glutathione peroxidase (GPX), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragments were immobilized on individual Hybond N membranes.


Figure 3: Transcriptional activity of GST- promoter-CAT fusion plasmid transiently transfected into KB and KB/BSO3 cells. GST- promoter-CAT gene fusion plasmid, p-2203 GST-CAT, p-130 GST-CAT, and p-80 GST-CAT were co-transfected with pSV2-beta-gal into KB and KB/BSO3 cells as described under ``Experimental Procedures.'' CAT activities were corrected for differences in transfection efficiency among the cell lines as estimated by beta-galactosidase activity and then normalized to corrected activity of RSV-CAT transfected cells. The plasmids were transfected in three independent experiments; each bar represents the mean of three experiments, and each error bar indicates S.D. from the mean. *, significantly different (p < 0.05) between KB and KB/BSO3 cells.


Southern Blot of GST- and -GCS

We next examined whether the increased expression of the -GCS gene was due to gene amplification by means of Southern blotting. As shown in Fig. 4A, genomic DNAs of KB and its BSO-resistant lines were digested with EcoRI and hybridized with the -GCS probe. The -GCS gene was amplified more than 4-fold in each BSO-resistant cell line in comparison with their parental KB cells. Therefore, increased -GCS mRNA levels appear to be due to gene amplification in these cell lines. In contrast, there were no changes in GST- gene in BSO-resistant cells when compared to parental KB cells (Fig. 4B).

Figure 4: Southern blot of the -GCS and GST- gene. DNA was extracted from KB and its BSO-resistant cell lines and digested with EcoRI. The digest was then electrophoresed, transferred to Hybond N, and hybridized with a -GCS probe (A) and a GST- probe (B). The molecular weight markers indicated by arrows are in kilobase pairs (kb).


Drug Sensitivities and Cellular Levels of Various Glutathione-related Enzymes in GST- cDNA Transfectants of KB/BSO3 Cells

We next examined further the relationship between decreased levels of GST- and increased levels of -GCS in resistant cell lines, and we examined whether collateral sensitivity to ethacrynic acid or alkylating agents was associated with decreased GST- levels. We established GST- cDNA transfectants of KB/BSO3 cells(21) . Two transfectants, BSO3-1 and BSO3-2, overexpressed GST- according to Western and Northern blots (Fig. 5, A and B). The -GCS mRNA level was increased about 5-fold in KB/BSO3 compared with KB cells, but both transfectants had levels of -GCS mRNA similar to levels in KB cells (Fig. 5C). The cellular mRNA levels of glutathione peroxidase and MGMT were also compared between KB/BSO3 and its GST- cDNA transfectants, but there were no differences in the mRNA levels of glutathione peroxidase and MGMT (data not shown). These transfection experiments suggest that overexpression of GST- modulates the expression of the -GCS gene in BSO3-1 and BSO3-2 cells.

Figure 5: Northern blot (A), immunoblot (B) of GST-, Northern blot of -glutamylcysteine synthetase (C) from KB, KB/BSO3, KB/BSO3-1, and KB/BSO3-2 cells. Total RNA (15 µg) extracted from KB, KB/BSO3, KB/BSO3-1, and KB/BSO3-2 was separated on a formaldehyde-agarose gel and transferred to a nitrocellulose membrane. Preparation of the probes and hybridization proceeded as described in the legend to Fig. 1(A and C). The immunoblotting of GST- proceeded as described in the legend to Fig. 1(F).


Table 2shows a comparison of the cellular sensitivity of KB, KB/BSO3, KB/BSO3-1, and KB/BSO3-2 to BSO, cisplatin, ACNU, melphalan, and ethacrynic acid. Transfection of GST- cDNA into KB/BSO3 cells restored wild type KB cell BSO sensitivity in the KB/BSO3-1 and -2 transfectants. These results suggest that GST- is involved in the modulation of cellular sensitivity to BSO. In addition, increased expression of GST- in both KB/BSO3-1 and KB/BSO3-2 restored the cellular resistance to melphalan, ACNU, and cisplatin to the levels similar to those observed in KB cells (Table 2). Drug resistance to ethacrynic acid in KB/BSO3 was also markedly increased in both GST- transfectants. By contrast, pSV2-neo transfectants without GST- overexpression showed almost similar sensitivities to various drugs as their parent KB/BSO3 cells (data not shown).

Comparison of Glutathione Levels in KB/BSO3 and Its GST- cDNA Transfectants in the Presence of BSO

Cellular GSH levels were compared among KB, KB/BSO3, KB/BSO3-1, and KB/BSO3-2 cells. KB/BSO3 cells had 2.0-fold more GSH than KB cells (Fig. 6). The GSH contents of KB/BSO3-1 or KB/BSO3-2 cells were similar to or lower than those of KB cells. The GSH levels of KB, KB/BSO3, BSO3-1, and KB/BSO3-2 cell lines were all reduced similarly to about 30% of the initial GSH levels by the addition 2.5 µM BSO (Fig. 6). However, the cellular GSH contents in KB/BSO3 cells were still higher than those in KB or the other two transfectants, and the GSH levels of KB/BSO3 cells in the presence of more than 2.5 µM BSO were comparable to those in KB or transfectants in the absence of BSO.

Figure 6: Total intracellular GSH levels in KB, KB/BSO3, KB/BSO3-1, and KB/BSO3-2 cells. Exponentially growing cells were exposed to various concentrations of BSO for 18 h, then the GSH content was determined. bullet, KB; , KB/BSO3; ▪, KB/BSO3-1; , KB/BSO3-2. Each point is the average of triplicate dishes. Bars, ±S.D.


DISCUSSION

We first selected three human cancer KB cell lines resistant to BSO, a synthetic amino acid inhibitor of -GCS(18, 19) . The expression of GST- was dramatically decreased with a concomitant increase of -GCS levels in the BSO-resistant cell lines. Three BSO-resistant cell lines, KB/BSO1, KB/BSO2, and KB/BSO3, were 10- to 13-fold more resistant to BSO than were parental KB cells. Cellular GSH levels are balanced through glutathione metabolism, and the critical reaction for GSH synthesis is catalyzed by -GCS and -glutamyl transpeptidase(1, 9) . These three BSO-resistant cell lines had similar levels of GSH (^2)and -GCS mRNA (Fig. 1). These findings suggest that the increased GSH and -GCS levels are associated with their BSO-resistant phenotypes. Nuclear run-on assay also showed that the -GCS mRNA transcribed in KB/BSO3 was higher than that of parent KB cells (Fig. 2). In addition, Southern blot analyses revealed that increased -GCS levels were due to gene amplifications in BSO-resistant cells. Thus, it is likely that the increased mRNA levels of -GCS are mainly attributable to gene amplification rather than activation of its promoter activity. Richman and Meister (8) have reported that both the synthesis and activity of -GCS are inhibited by GSH through a feedback mechanism.

In GSH metabolism, the cellular level of GSH is also controlled by glutathione peroxidase and GST(9) . The expression of GST-, but not of glutathione peroxidase, was markedly reduced specifically in all the BSO-resistant cell lines (Fig. 1). Nuclear run-on assay (Fig. 2) and GST- promoter-CAT experiment (Fig. 3) revealed that the decreased expression of GST- gene in BSO-resistant cells was due to reduced transcriptional activity. Furthermore, transient transfection assays of GST- promoter-CAT (Fig. 3) suggest that the cis-regulatory elements between -130 bp and -80 bp from the initiation site might be at least partially responsible for decreased GST- expression in BSO-resistant cells. As Southern blots revealed no deletion and rearrangement of the GST- gene in BSO-resistant cells (Fig. 4B), we conclude that decreased expression of GST- gene is mainly due to altered transcriptional regulation. Human GST- promoter sequences contain no binding sites of known regulatory factors between -130 and -80 from the initiation site(38) . In mouse, rat, and human GSTs gene, a TPA-responsive element or AP-1 binding site is the common motif of their promoter regions(23, 46, 47, 48, 49, 50) . The mouse GST-Ya subunit gene is controlled by an EpRE enhancer which contains two AP-1-like binding sites(46, 47) . Bergelson et al.(48) have recently reported that Fos-Jun heterodimeric complex (AP-1)-mediated transcription activation of the rat GST-Ya gene acts through a common mechanism involving the production of reactive oxygen species and the depletion of GSH. Rat GST-P, which is a homologue of human GST-, contains two TPA-responsive elements in its promoter region: one is in the enhancer element GPE1 (GST-P enhancer I) at the position of -2.5 kilobases, and the other is at -61 bp upstream of the GST-P transcriptional start point(49) . TPA treatment stimulated endogenous GST-P transcription (49) , but Morimura et al.(50) have reported that involvement of Fos-Jun in rat GST-P gene expression is less likely in rat hepatoma cells. Morrow et al.(38) revealed that neither TPA treatment nor co-transfection of Fos-Jun expression vectors induced human GST- promoter activity. There appears to be no consistent regulatory mechanism for GST gene expression, in mammalian cells. Further study is required to understand the molecular mechanism underlying defective GST- gene expression in our BSO-resistant cell lines.

Furthermore, the introduction of GST- cDNA into KB/BSO3 cells resulted in a decrease of the GSH levels (Fig. 6). GSTs play an important role in detoxification by conjugating many xenobiotics and other hydrophobic/electrophilic compounds with a concomitant decrease in GSH levels(51) . Consistent with this notion, GST- may play a critical role in the balance of cellular GSH levels and also in the acquisition of BSO-resistant phenotypes in KB cells. On the other hand, cellular GSH levels were efficiently depleted when incubated with various doses of BSO up to 5 µM (Fig. 6). KB and KB/BSO3 showed similar percent depletion of GSH levels in response to BSO treatment, suggesting that the membrane permeability of BSO is not blocked in the resistant cell line.

BSO-resistant cell lines were collaterally sensitive to alkylating agents such as nitrosourea (ACNU) or melphalan as well as ethacrynic acid (Table 1). The expression of the MGMT gene often confers resistance to the toxic effects of alkylating agents in mammalian cells (31, 45, 52) . However, all BSO-resistant cell lines had similar levels of MGMT, suggesting that this enzyme is probably not involved in the collateral sensitivity to alkylating agents. The transfection of KB/BSO3 cells with GST- cDNA, however, made them more than 3-fold resistant to ethacrynic acid than the wild type KB cells (Table 2). Transfection with the GST- gene confers upon human breast cancer MCF-7 cells drug resistance to ethacrynic acid (23) and mouse NIH3T3 cells(24) . These findings consistently support the notion that cellular sensitivity to ethacrynic acid, a diuretic substrate which is a substrate for GST(53) , is closely correlated with GST- levels.

Cisplatin is an effective anticancer agent, which functions by interacting with its target DNA through interstrand or intrastrand cross-links. Drug resistance to cisplatin is mediated through pleiotropic mechanisms, including protection by GSH compounds, drug transport, DNA repair, and metallothionein(54, 55, 56) . We also demonstrated that the acquisition of cisplatin resistance is due to elevated GST- levels(20, 21) , decreased intracellular accumulation of the drug(57, 58) , decreased levels of DNA topoisomerase I(44) , and DNA repair(59) . The cellular GSH levels often influence drug resistance to cisplatin as well as to alkylating agents(10) . Increased cellular GST- levels are closely associated with resistance to cisplatin in Chinese hamster ovary cells (20, 21) or in patients with gastric cancer(60) , but not with resistance to cisplatin in human breast cancer cells or mouse fibroblasts(23, 24) . In this study, KB/BSO3 cells showed a slight collateral sensitivity, whereas relative resistance was restored to 1.2 by introducing the GST- gene (Table 2). Drug sensitivity to cisplatin in human cancer KB cells is thus only slightly if at all modulated by the GST- levels. On the other hand, Godwin et al.(25) have reported that cisplatin resistance in human ovarian cancer cells is closely associated with increased cellular -glutamyl transpeptidase and -GCS levels. Their cisplatin-resistant ovarian cancer cell lines show very high degrees of drug resistance, being 30- to 1000-fold more resistant than their parental cells. However, all cell lines had similar GST activities(25) . The -GCS levels were 3- to 5-fold higher in BSO-resistant cell lines than in KB cells, whereas the levels of -glutamyl transpeptidase were similar among our resistant and sensitive cell lines (Fig. 1). BSO-resistant cell lines were not cross-resistant, but rather collaterally sensitive, to cisplatin, suggesting that increased -GCS levels do not directly influence the sensitivity to cisplatin.

Among the BSO-resistant cell lines with increased levels of -GCS mRNA, cellular GST- mRNA levels were decreased. Transfection of GST- into BSO-resistant KB/BSO3 cells restored -GCS mRNA, GSH contents, and sensitivity to BSO and other drugs to levels similar to those of wild type KB cells (see Table 3). Our present findings suggest that GST- levels might be obligatorily coupled with -GCS levels in human cancer KB cells. However, this hypothesis comes from our experiments with GST- transfectants, which were established during incubation for about 1 month in the absence of BSO. One could argue that -GCS gene amplification in KB/BSO lines is unstable, resulting in rapid loss during selection of the transfectants in the absence of BSO. However, both BSO-resistant phenotype and -GCS amplification had been stably maintained for 1 month in KB/BSO3 cells in the absence of BSO, and pSV2-neo transfectants of KB/BSO3 without overexpression of GST- gene showed similar levels of drug sensitivities and -GCS gene amplification as those of KB/BSO3 cells.^2 In our present study, -GCS gene expression was not determined in KB/BSO3 cells immediately after the exogenous GST- expression plasmid was introduced. Furthermore, it remains unknown whether overexpression of -GCS gene could also affect GST- gene expression. The hypothesis whether cellular GST- levels could directly modulate expression of -GCS gene is speculative pending further study. To examine whether this selective reduction of GST- mRNA levels in BSO-resistant cells is a common phenomenon, further isolation and characterization of BSO-resistant variants from other cell types will be required.

FOOTNOTES

*
This study was supported by a grant-in-aid for scientific research from Ministry of Education, Science and Culture, Japan, and in part from the Yasuda Memorial Medical Grant for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, Kyushu University School of Medicine, Maidashi 3-1-1, Fukuoka 812-82, Japan. Tel.: 81-92-641-1151 (Ext. 3351); Fax: 81-92-632-4198.

(^1)
The abbreviations used are: -GCS, -glutamylcysteine synthetase; BSO, DL-buthionine-[S,R]-sulfoximine; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; ACNU, 1-(4-amino-2-methyl5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride; cisplatin, cis-platinum(II)diammine dichloride; MGMT, O^6-methylguanine methyltransferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; bp, base pair(s).

(^2)
A. Yokomizo, K. Kohno, M. Wada, M. Ono, and M. Kuwano, unpublished data.

ACKNOWLEDGEMENTS

We thank Hiroshi Hayakawa and Takanori Nakamura in our laboratory for fruitful discussions and also Akiko Mori for preparing this manuscript.

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