Regulation of γ-glutamylcysteine synthetase regulatory subunit (GLCLR) gene expression: identification of the major transcriptional start site in HT29 cells

Biochimica et Biophysica Acta 1446 (1999) 47^56 www.elsevier.com/locate/bba

Regulation of Q-glutamylcysteine synthetase regulatory subunit (GLCLR) gene expression: identi¢cation of the major transcriptional start site in HT29 cells Diane C. Galloway, David G. Blake 1 , Lesley I. McLellan * Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK Received 14 December 1998; received in revised form 26 April 1999; accepted 4 May 1999

Abstract Q-Glutamylcysteine synthetase (GCS) is of major importance in glutathione homeostasis. The GCS heterodimer is composed of catalytic (heavy subunit, GCSh ) and regulatory (light subunit, GCSl ) subunits. Regulation of the human GCSl subunit gene (GLCLR) expression was studied as GCSl has a critical role in glutathione synthesis. The minimal basal expression of GLCLR was found to be mediated by a region between nt 3205 and 3318. The major transcriptional start site in HT29 cells was located within this region at nt 3283. A region between nt 3411 and 3447 was identified as having a potential involvement in the negative regulation of GLCLR expression. In order to study the transcriptional regulation of GCSl by oxidant stress, HepG2 cells were treated with sodium nitroprusside (SNP). SNP (1.5 mM) was found to increase glutathione levels by 2-fold, as well as GCS activity by 6-fold. This is accompanied by a co-ordinate increase in the levels of the both the GCSl and GCSh subunits, each by approximately 2-fold. The transcriptional activity of the GLCLR gene was increased by approximately 2.5-fold in SNP-treated cells. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Glutathione; Q-Glutamylcysteine synthetase; GLCLC; GLCLR; Sodium nitroprusside

1. Introduction Glutathione, an abundant intracellular tripeptide thiol, has a number of fundamental roles, including

Abbreviations: CAT, chloramphenicol acetyltransferase ; GCS, Q-glutamylcysteine synthetase; GCSh , GCS heavy subunit; GCSl , GCS light subunit; GLCLC, GCSh gene; GLCLR, GCSl gene; Inr, Initiator; NO, nitric oxide; SNP, sodium nitroprusside; tBHQ, tert-butylhydroquinone * Corresponding author. Fax: +44-1382-663999; E-mail; [email protected]. 1 Present address: Cyclacel Ltd., Dundee Incubator Unit, James Lindsay Place, Dundee DD1 5JJ, UK.

detoxi¢cation of xenobiotics, maintenance of the redox status and cellular protection against reactive oxygen species [1]. The regulation of glutathione homeostasis is therefore of considerable importance, where the de novo biosynthesis of glutathione is likely to play a critical role [2]. Glutathione is synthesised by two ATP-dependent reactions, catalysed by Q-glutamylcysteine synthetase (GCS) and glutathione synthetase [2]. The rate-limiting reaction is catalysed by GCS, although the availability of cysteine is also proposed to be a rate-limiting factor [3]. GCS is a heterodimer composed of catalytic (heavy subunit, GCSh ) and regulatory (light subunit, GCSl ) subunits [4,5]. The GCSl subunit has an important role in regulating GCS activity, by modulating the

0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 0 7 3 - 1

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Km for glutamate and the feed-back inhibition by glutathione [6,7]. Glutathione contributes to the intracellular antioxidant defence system and thus plays a key role in cellular protection against oxidative stress [8]. Glutathione homeostasis can be regulated by oxidative stress, and several studies have reported that chemicals which generate oxidative stress can result in increased glutathione levels, GCS activity and GCSh expression [8^10]. By contrast, few studies have investigated the e¡ect of oxidative stress on the expression of the GCSl subunit. Recent reports, however, have demonstrated that certain chemical agents co-ordinately increase the expression of the GCSh and GCSl subunits [11^14]. The increases in levels of the GCS subunits has been attributed to an increase in the transcription rates of the GCSh (GLCLC) and GCSl (GLCLR) subunit genes [11^ 14]. Previous analyses of the GLCLR gene in our laboratory have demonstrated that the increased expression of the GCSl subunit by tert-butylhydroquinone (tBHQ) is attributable to an increase in the transcriptional activity of the GLCLR gene [15,16]. Within the present study, we have analysed GLCLR 5P-regions which may regulate basal expression as well as the e¡ect of oxidative stress, induced by the nitric oxide (NO) donor sodium nitroprusside (SNP), on glutathione homeostasis and on the transcriptional activity of the GLCLR gene in HepG2 cells.

of the GCSl cDNA [15] (Fig. 1C). Exonuclease III digestion from the 3P-end of pDGCAT1 generated pDGCAT1.1 and pDGCAT1.2 which contain 5Pfragments upstream of nt 3256 and 3411, respectively. MluI^XbaI fragments (with the XbaI site being contained in the multiple cloning site of the pCAT vector, and the MluI site being within the GLCLR gene at nt 3878) of pDGCAT1.1 and pDGCAT1.2 were substituted with the corresponding region of pDGCAT4, generating pDGCAT4.1 and pDGCAT4.2, respectively. The GLCLR fragment between nt 3411 and 31638 of pDGCAT1.2 was excised as an AccI^XbaI fragment and subcloned into the pCAT-Promoter vector, which contains the SV40 promoter, generating pDGCATP1638. The region between nt 3324 and 3447 was ampli¢ed by PCR and subcloned into pCAT-Promoter, generating pDGCATP324/447. Site-directed mutagenesis was performed [18] on the initiator (Inr) element in pDGL5v205 [15], which contains an approximately 1.7-kb GLCLR fragment in pBluescript II SK(+). An MluI^XbaI fragment (0.67 kb) containing the mutated bases was substituted into the corresponding region in pDGCAT4, generating pDGCAT4mI. Mutated products and PCR products were sequenced using a doublestranded DNA cycle sequencing kit (Gibco-BRL, Paisley, Renfrewshire, UK)

2. Materials and methods

The human hepatocyte carcinoma cell line HepG2 was maintained in Dulbecco's modi¢ed Eagle's medium with 862 mg/l Glutamax I, 110 mg/l sodium pyruvate and 1 g/l glucose (Gibco-BRL) supplemented with 10% (v/v) heat-inactivated foetal calf serum, 50 IU/ml penicillin and 50 Wg/ml streptomycin. The human colon adenocarcinoma cell line HT29 was maintained in Dulbecco's modi¢ed Eagle's medium with 110 mg/l sodium pyruvate (GibcoBRL), supplemented with 10% (v/v) heat-inactivated foetal calf serum, 50 IU/ml penicillin, 50 Wg/ml streptomycin, and 2 mM L-glutamine. All cells were grown at 37³C in a humidi¢ed air/CO2 (19:1) atmosphere. HepG2 cells were seeded at a density of 2.5U106 per 10 cm plate 24 h prior to chemical treatment. Cells were treated for 18^20 h with 0.75, 1 or

2.1. Recombinant plasmids The reporter vectors pCAT-Basic, pCAT-Promoter and pCAT-Control were purchased from Promega, Southampton, UK. The GLCLR reporter constructs pDGCAT1 and pDGCAT4 contain an approximately 6 and 0.8 kb fragment (each upstream of nt 3205) of the GLCLR gene, respectively, upstream of the promoterless CAT reporter gene [15]. Unidirectional digestion with exonuclease III [16,17] from the 5P-end of pDGCAT4 produced a series of deletion constructs, designated pDGCAT530, pDGCAT447 and pDGCAT318. The number refers to the GLCLR nt at the 5P-end of the construct, with all numbers relative to the A of the initiation codon

2.2. Cell culture and treatment

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Fig. 1. Regulation of the basal expression of the GLCLR gene. A schematic representation of a series of deletion constructs of pDGCAT4 is shown in A, where ¢lled boxes denote GLCLR DNA and open boxes denote CAT reporter DNA. The nt positions are numbered relative to the A residue of the initiation codon of the GCSl cDNA. B shows the CAT activity of HepG2 cells transiently transfected with the series of deletion reporter constructs (as indicated in A). Transfections were performed in triplicate and the values are the mean þ S.E.M., expressed as a percentage of the CAT activity of pCAT-Control. Where error bars appear to be absent, the S.E.M. was too small to be shown on the histogram. C shows the sequence of the GLCLR promoter region, indicating the relative positions of the ¢rst nucleotide of the reporter constructs and depicting potential binding sites. The AP-1 site and ARE are depicted in bold, whereas the 42-bp sequence shown to be important in tBHQ-mediated induction [16] is underlined. The region between nt 3447 and 3411 which is likely to have a negative e¡ect on transcription is highlighted as white-on-black. The upstream region between nt 3530 and nt 3447 which may also have a role in modulating basal expression is outlined with a hatched line. The region required for minimal basal promoter activity is located between 3318 and 3205. This is indicated by the grey shading. The major transcriptional start site in HT29 cells is indicated by the bold arrow. Nucleotides are numbered relative to the initiation codon (doubly underlined).

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1.5 mM SNP. Cells were lysed in 10 mM sodium phosphate, pH 7.2, containing 2 mM MgCl2 and 1 mM EDTA pH 8.0 by repeated freeze^thaw cycles. Insoluble cell debris was removed by centrifugation. Transient transfection of HepG2 cells was carried out as described previously [15]. Transfected cells were treated with 1.5 mM SNP in DMSO, or an equal concentration of DMSO as control for 18^20 h. L-Galactosidase and chloramphenicol acetyltransferase (CAT) assays were performed as described previously [15]. 2.3. Analytical Protein concentrations in cell lysates were determined by the method of Bradford [19], modi¢ed for use on a Cobas Fara centrifugal analyser (Roche Diagnostics, Welwyn Garden City, Herts., UK). Up to 29 samples (each of 8 Wl, and with a diluent volume of 30 Wl H2 O) were simultaneously mixed and incubated at 37³C with 320 Wl Coomassie brilliant blue G250 solution (0.01% (w/v) in distilled water containing 5% (v/v) ethanol and 8.5% (v/v) phosphoric acid) for 180 s. Absorbance at 595 nm was measured, and the protein concentration was calculated from a standard curve generated using bovine serum albumin at concentrations between 62.5 and 500 Wg/ml. Glutathione concentrations were measured by the method of Tietze [20], also adapted for use on the Cobas Fara centrifugal analyser. Up to 29 samples (each of 10 Wl, and with a diluent of 25 Wl H2 O) were pipetted in parallel with 150 Wl of reagent 1 (0.34 mM NADPH, 1 mM 5,5P-dithio-bis(2-nitrobenzoic acid) in 150 mM sodium phosphate bu¡er, pH 7.5, containing 7.5 mM EDTA) and mixed in the reaction cuvettes for 60 s at 30³C. The reaction was started by the addition of 50 Wl (1 unit) bakers yeast glutathione reductase (Sigma, Poole, Dorset), previously diluted in 150 mM sodium phosphate bu¡er, pH 7.5, containing 7.5 mM EDTA, and the change in absorbance at 412 nm measured over 30 s at 30³C. Glutathione concentrations in the samples were calculated from a standard curve. GCS activity was measured by an adaptation of the method of Hamel et al. [21]. Brie£y, 20 Wl of cell extract (containing 100 Wg protein) was added to 80 Wl assay bu¡er (200 mM Tris/HCl, pH 7.4, con-

taining 20 mM ATP, 15 mM MgCl2 , 0.2 mM EDTA, 120 mM glutamic acid) and incubated at 37³C for 5 min. The reaction was started by the addition of 50 Wl 20 mM cysteine, and allowed to proceed for 20 min at 37³C, following which 50 Wl 200 mM sulphosalicylic acid was added to terminate the reaction and precipitate protein. Reaction mixtures were chilled on ice for 10 min and the precipitated protein pelleted by centrifugation on a bench-top microcentrifuge. A portion (100 Wl) of the supernatant was added to 80 Wl 0.25 M N-ethyl morpholine/25 mM KOH, to adjust the pH to between pH 8.0 and 9.0, followed by the addition of 20 Wl monobromobimane solution (25 mM in acetonitrile). The reaction of monobromobimane with the non-protein thiol groups was allowed to proceed in the dark for 20 min at room temperature before termination by the addition of 100 Wl sulphosalicylic acid. Portions (50 Wl) of the reaction mixtures were analysed by reverse phase HPLC (Hewlett Packard 1050 automated system, Hewlett Packard, UK) as described [21]. 2.4. Western blot analysis Antibodies raised against peptides corresponding to regions of the GCSh or GCSl subunits [15] were used at a dilution of 1:500 for Western blot analysis. Proteins were resolved on SDS-polyacrylamide gels containing 10% (w/v) acrylamide and electro-transferred to nitrocellulose membrane [22]. Rat kidney cytosol, which contains relatively high levels of each of the GCS subunits, was used as a standard to con¢rm the identity of the bands. The relative intensities of bands on autoradiographs were estimated by scanning densitometry. Loading and transfer of samples was assessed by staining the nitrocellulose membranes after transfer with 0.2% (w/v) Ponceau S (3-hydroxy-4-[2-sulpho-4-(4-sulphophenylazo)-phenylazo]-2,7-naphthalenedisulphonic acid) in distilled water containing 3% (w/v) trichloroacetic acid and 3% (w/v) sulphosalicylic acid. Membranes were incubated with the Ponceau S solution for 1 min at room temperature to visualise transferred protein. 2.5. Primer extension analysis Total RNA from HT29 or HepG2 cells was pre-

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pared using the method of Chomczynski and Saachi [23]. An oligonucleotide primer complementary to nt 3266 to 3427 of the GLCLR gene, was labelled at the 5P-end with [Q-32 P]ATP using T4 polynucleotide kinase (Promega). The labelled oligonucleotide and 10 Wg of total RNA were precipitated, and subsequently hybridised in 10 mM Tris/HCl pH 8.0, 300 mM KCl, 0.2 mM EDTA pH 8.0 at 45³C for 16^18 h. The reaction mixture was chilled on ice prior to the addition of 10 Wl of 5Ureaction bu¡er (0.25 M Tris/HCl pH 8.3, 15 mM MgCl2 , 50 mM DTT). The extension reaction was initiated by the addition of 10 Wl of 2.5 mM of dATP, dGTP, dCTP, dTTP (Pharmacia, Uppsala, Sweden), and the primer extended by M-MLV reverse transcriptase (200 U) (Gibco BRL) at 37³C for 45 min. The extended single stranded DNA products were extracted with phenol/chloroform (1:1), precipitated with ethanol, and resuspended in loading bu¡er (95% (v/v) formamide, 10 mM EDTA pH 8.0, 0.1% (w/v) Bromophenol blue, 0.1% (w/v) xylene cyanol). The products were analysed on an 8% (w/v) polyacrylamide/8 M urea sequencing gel, and visualised by autoradiography on X-ray ¢lm (Fuji, Tokyo, Japan). 2.6. Statistical analysis Statistical analyses were performed using the Student's unpaired t-test. Values were considered to be signi¢cant if P 6 0.05. 3. Results 3.1. Basal expression of the GLCLR gene We have isolated previously approximately 6 kb of the 5P-£anking region of the GLCLR gene, and revealed that the region important for basal promoter activity is located between nt 31007 and 3205 [15]. The basal expression of the GLCLR gene has been reported to be regulated by a proximal AP-1 site [16,24]. Within the present study, we analysed a series of GLCLR deletion reporter constructs, in order to identify the 5P-GLCLR region which interacts with the basal promoter machinery. The CAT activities of pDGCAT4, pDGCAT530, pDGCAT447 and pDGCAT318 which contain GLCLR fragments

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downstream of nt 31007, 3530, 3447 and 3318, respectively, were determined in HepG2 cells. All of these constructs exhibit CAT activity (Fig. 1), including pDGCAT318, indicating that the minimal region required for basal promoter activity is located between nt 3318 and 3205. The CAT activity of pDGCAT447 is approximately 60% lower than the CAT activity of pDGCAT530 (P 6 0.001), indicating that a region important for modulating basal expression of GLCLR is located between nt 3447 and 3530. The position of this region relative to other potential regulatory sites within the GLCLR promoter is shown in Fig. 1C. Interestingly, the promoter activity of pDGCAT318 is increased approximately 2-fold compared with pDGCAT447 (P 6 0.001). The activity of pDGCAT318, however, is approximately 35% lower than pDGCAT530 and pDGCAT4 (P 6 0.025) which may be partly a consequence of the absence of the proximal AP-1 site (Fig. 1C) which we and others have shown to be involved in regulating basal promoter activity [16,24]. The region between nt 3205 and 3256 was also found to be important for basal expression of the GLCLR gene, as the promoter activity of pDGCAT4.1, which lacks this region, is approximately 50% lower than the promoter activity of pDGCAT4 (P 6 0.005) (Fig. 1). Removal of the region between nt 3205 to 3411 almost abolishes the transcriptional activity of the GLCLR reporter construct, supporting the proposal that the region downstream of nt 3411, and between nt 3318 and 3205 is essential for basal expression of the GLCLR gene. As the promoter activity of pDGCAT447 is lower than pDGCAT318, this implies that a negative regulatory region may be present within the region between nt 3447 and 3318. We have demonstrated previously that an approximately 6.0 kb GLCLR region upstream of nt 3411 can repress the promoter activity of a heterologous promoter [16]. To investigate the potential negative regulatory characteristics of 5P-regions of the GLCLR gene, the e¡ect of GLCLR fragments on the activity of the SV40 promoter, contained in the pCAT-Promoter vector, were analysed. The regions between nt 3411 and 31638, and between nt 3324 and 3447 were subcloned into the pCAT-Promoter vector generating pDGCATP1638 and pDGCATP324/447. The promoter activities of the two constructs were approx-

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Table 1 Negative regulation of GLCLR promoter activity Reporter construct

CAT activity (% of pCAT-Promoter)

pDGCATP1638 pDGCATP324/447

23.0 þ 3.9 31.1 þ 0.7

The CAT activity of HepG2 cells transiently transfected with pDGCATP1638 and pDGCATP324/447 was measured. Transfections were performed in triplicate and the values are the mean þ S.E.M., expressed as a percentage of the CAT activity of the pCAT-Promoter vector.

imately 70% lower than the promoter activity of pCAT-Promoter (P 6 0.001) (Table 1). This implies that a region within these GLCLR fragments can repress the basal promoter activity of the heterologous promoter. As the region between nt 3411 and 3447 is present within these two constructs and pDGCATP1.2 [16], this supports the hypothesis that a negative regulatory region is likely to be located between nt 3411 and 3447; however, additional upstream regions may also contain negative regulatory elements. Collectively, the data suggest that the lower CAT activity of pDGCAT447 is mediated by the presence of a negative regulatory element, which is likely to contribute to the lower promoter activity of this reporter construct. Fig. 1C shows the position of the putative negative regulatory region relative to other potential regulatory regions within the GLCLR promoter.

As the region between nt 3205 and 3318 was found to be essential for the basal promoter activity of the GLCLR gene, this region was analysed by primer extension analysis to determine the transcription start site. These experiments were performed using RNA from human colon adenocarcinoma HT29 cells and human liver hepatocellular carcinoma HepG2 cells. In HT29 cells, several minor and one major reverse transcribed products were detected, with the major transcription start site being located at the A residue at nt 3283 (Fig. 2). The position of this start site relative to other binding sites is indicated in Fig. 1C. Nucleotide 3283 corresponds to nucleotide 3282 in the sequence reported by Moinova and Mulcahy [24]. Analysis of the neighbouring 5P-region of the GLCLR gene revealed that a TATA box, which accurately positions the transcription start site in many mammalian promoters, is not located upstream of this transcription start site in the GLCLR gene. We were unable to identify de¢nitively a major transcriptional start site in HepG2 cells (data not shown). A second type of core promoter, called an initiator (Inr) is frequently found in TATA-less genes [25]. The consensus sequence for Inr elements is Py,Py,A‡1 ,N,T/A,Py,Py [25]. A sequence which resembles the Inr element is present in the GLCLR gene at nt 3269 to 3263. The A residue, at nt 3267, corresponds to one of the potential transcriptional start sites of the GLCLR gene which we observed in HepG2 cells (data not shown). To analyse the importance of the potential Inr, the element was mutated in the reporter construct pDGCAT4, from TCAGTTT to TCGGGT, generating pDGCAT4mI. Transient transfection of HepG2 cells with pDGCAT4 and pDGCAT4mI, however, revealed that the promoter activity of pDGCAT4mI was not Table 2 Analysis of a potential Inr element in the GLCLR gene

Fig. 2. Transcriptional start site of the GLCLR gene. Primer extension analysis was performed with total RNA isolated from HT29 cells and an oligonucleotide complementary to nt 3427 to 3266. The reverse transcribed products were separated by PAGE, and visualised by autoradiography. The arrow denotes the position of the major transcriptional start site, at nt A, position 3283. The DNA sequence of the proximal region is annotated.

Reporter construct

CAT activity (% of pCAT-Control)

pDGCAT4 pDGCAT4mI

90.2 þ 1.0 111.6 þ 0.5

The CAT activity of HepG2 cells transiently transfected with the reporter constructs pDGCAT4 and pDGCAT4mI was measured. Transfections were performed in triplicate and the values are the mean þ S.E.M., expressed as a percentage of the CAT activity of pCAT-Control.

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Fig. 3. The e¡ect of sodium nitroprusside on glutathione levels and GCS activity in HepG2 cells. HepG2 cells were treated with 0.75 mM, 1 or 1.5 mM SNP for 18^20 h. Cells were harvested and the glutathione concentration (A) and GCS activity (B) determined in cell lysates. Experiments were performed in triplicate and the values are the mean þ S.E.M. Where error bars appear to be absent, the S.E.M. was too small to be shown on the histogram. Signi¢cance is indicated as follows: **P 6 0.001; 3 P 6 0.01; *P 6 0.025.

signi¢cantly lower than pDGCAT4 (Table 2). This data suggests that in HepG2 cells, the putative transcriptional start site at nt 3267 is either not utilised for transcription of GLCLR, or that it is not the sole transcriptional start site and that other regions, apart from the Inr element, are likely to control accurate transcription. 3.2. Inducible expression of the GLCLR gene We have shown previously that tBHQ increases glutathione levels in HepG2 cells, which may be a consequence of a co-ordinate increase in the mRNA and protein levels of the GCSh and GCSl subunits [15]. Evidence suggests that the increase in levels of the GCSl polypeptide is a result, at least in part, of increased transcriptional activity of the GLCLR gene [16]. As oxidative stress has been reported previously to regulate GSH homeostasis, we have analysed the e¡ect of SNP, a NO generating compound which induces oxidative stress, in HepG2 cells. We observed an approximately 2-fold increase in glutathione levels from 189.4 þ 39.2 to 360.4 þ 85 nmol/mg protein in HepG2 cells treated with 1.5 mM SNP (P 6 0.025) (Fig. 3A). The increase in glutathione levels was paralleled by an approximately 6-fold increase in GCS activity from 317.8 þ 22.5 to 1959.2 þ 423.2 pmol/min/mg protein

(P 6 0.01) (Fig. 3B). Western blot analysis showed that the levels of the GCSl polypeptide are also increased in SNP-treated cells by approximately 2-fold (Fig. 4). Increased levels of GCSh were also observed. In order to determine whether this was likely to be a consequence of transcriptional activation of the GLCLR gene, HepG2 cells were transiently transfected with the reporter constructs, pDGCAT1, which contains approximately 6 kb of the 5P-£anking region of GLCLR upstream of the CAT reporter gene [15] and pDGCAT4. pDGCAT4 has higher transcriptional activity than pDGCAT1, which we have noted previously [15], and an increase in

Fig. 4. Induction of GCS subunits in SNP-treated cells. HepG2 cells were treated with SNP for 18^20 h, and cell lysates prepared. Protein (20 Wg) was resolved by SDS-PAGE and electrotransferred to nitrocellulose membrane. Western blot analysis using antisera raised against peptides corresponding to a region of the GCSl subunit or the GCSh subunit was performed. Lanes were loaded with extracts from cells treated as follows: lane 1, no treatment; lane 2, 0.75 mM SNP; lane 3, 1 mM SNP; and lane 4, 1.5 mM SNP.

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Fig. 5. Transcriptional activity of GLCLR in SNP-treated cells. The CAT activity of HepG2 cells transiently transfected with the reporter constructs pDGCAT1 or pDGCAT4, was measured in control and SNP-treated cells. Transfections were performed on three separate occasions and the values are the mean þ S.E.M. Where error bars appear to be absent, the S.E.M. was too small to be shown on the histogram. Values are expressed as a percentage of the CAT activity of pCATControl.

CAT activity of 2.5-fold (P 6 0.005) and 2.2-fold (P 6 0.01) was found for pDGCAT1 and pDGCAT4, respectively, in SNP-treated cells (Fig. 5). This is a similar level of induction to that mediated by tBHQ [15]. These data suggest that increased transcription of the GLCLR gene appears to contribute to the increased expression of the GCSl subunit in response to SNP, and that this is likely to be mediated by a region of DNA downstream of nt 31007. 4. Discussion The basal expression of the GLCLR gene has been reported previously to be regulated by an AP-1 site in the proximal region of the GLCLR gene [16,24]. AP-1 has also been found to regulate the constitutive expression of the GLCLC gene [26,27]. During the present study, we have identi¢ed additional regions which are important for the basal expression of the GLCLR gene. Analysis of a series of deletion constructs revealed that the basal promoter machinery is

likely to interact with the region between nt 3205 and 3318. These results are consistent with a recent report which showed that a GLCLR region between nt 3242 and 3344 is important for minimal basal expression of the GLCLR gene [24]. We have identi¢ed the major transcriptional start site of the GLCLR gene in HT29 cells which is located within this region at nt 3283. Sequence analysis revealed that, unlike the GLCLC gene, a traditional TATA box is not located in the region proximal to the transcriptional start site [28]. However, a sequence which resembles the Inr element consensus sequence, which correctly positions the transcriptional start site in many TATA-less genes [25], was identi¢ed between nt 3263 to 3269. The A residue, at nt 3267, would correspond to the A‡1 residue within the Inr consensus sequence (Py,Py,A‡1 ,N,T/A,Py,Py), and this was identi¢ed as a putative transcriptional start site in HepG2 cells. Mutation analysis of the Inr element, however, did not reduce the level of transcription from the GLCLR reporter construct in HepG2 cells. This implies that, in HepG2 cells, alternative transcriptional start sites may be functional within GLCLR. Other mechanisms apart from those involving a TATA box or an Inr element are likely to contribute to accurate positioning of the basal promoter machinery in GLCLR. Regions capable of negatively regulating the expression of the GLCLR gene were also identi¢ed. The region between nt 3411 and 3447 (with the DNA sequence 5P-CCCGCCGCGCACCACCCGTCGCCACGCCCGCCGCAGC-3P) is proposed to contain a negative regulatory sequence, as this region is the only overlapping fragment present in the three GLCLR fragment which are capable of repressing the promoter activity of a heterologous promoter. The putative negative regulatory element within this region is also likely to contribute to the lower transcriptional activity of the reporter construct pDGCAT447 compared with pDGCAT318. As the promoter activity of pDGCAT530, which would also contain the repressor element, is higher than pDGCAT447, this suggests that the e¡ect of the negative repressor may be in£uenced by additional upstream regions. Furthermore, the removal of a positive regulatory element may also contribute to the lower promoter activity of pDGCAT447.

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The recent studies of Moinova and Mulcahy [24] have also shown the presence of additional regulators of basal expression of GLCLR. As well as showing regulation of basal expression by an AP-1 site, this group demonstrated that a region of DNA between nt 3712 and 3344 has a positive regulatory e¡ect on basal expression. This, together with the data generated in the present study, suggests that both positive and negative modulators of constitutive transcription may exist downstream of nt 3712 in the promoter of GLCLR (Fig. 1C). Treatment of HepG2 cells with SNP was found to increase glutathione levels. SNP is capable of generating an oxidative stress, and other agents which can produce oxidative stress have been shown previously to modulate glutathione homeostasis in human cells by regulating the de novo biosynthesis of glutathione [13^15]. We found that the increase in glutathione levels was accompanied by an increase in GCS activity, with increased levels of both the GCSh and GCSl polypeptides being observed. Co-ordinate upregulation of the expression of GCSh and GCSl subunits has also been found previously by tBHQ, diethylmaleate and L-naphtho£avone, and is proposed to be the result of increases in the transcriptional activity of the GLCLC and GLCLR genes, as well as an increase in the stability of the mRNA transcripts [11,14^16,24]. The increase in levels of the GCSl subunit in HepG2 cells by tBHQ was found to be associated with an increase in the transcriptional activity of the GLCLR gene [15]. The increase in levels of the GCSl subunit in SNP-treated cells is also likely to be a result of an increase in the transcriptional activity of the GLCLR gene, as expression of the reporter constructs pDGCAT4 and pDGCAT1 are increased by 2.2-fold and 2.5-fold, respectively. The co-ordinate regulation of the expression of the two subunits is of importance as the GCSl subunit has a critical regulatory role; it modulates the catalytic e¤ciency of the heterodimer by regulating the Km of glutamate and altering the sensitivity of feedback inhibition by glutathione. Furthermore, it has been shown previously that in the absence of increased levels of GCSh , over-expression of the GCSl polypeptide in HeLa cells results in increased GCS activity [29]. An increase in GCSl together with GCSh may be essential for the observed increases in glutathione levels and

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GCS activity in HepG2 cells treated with pro-oxidants. In conclusion, we have demonstrated that SNP regulates expression of GCS subunits and glutathione levels in HepG2 cells. The increased levels of the GCSl polypeptide appear to be attributable to an increase in the transcriptional activity of the GLCLR gene. We have identi¢ed the major transcriptional start site of the GLCLR gene in HT29 cells and characterised proximal regions important for basal expression. Acknowledgements We gratefully acknowledge the Wellcome Trust (D.C.G., Grant 044446/Z/95/Z) and the Association for International Cancer Research (D.G.B.) for ¢nancial support.

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