Glutamyl transpeptidase and glutathione biosynthesis in non ...

Carcinogenesis vol.23 no.4 p.669, 1999

Erratum γ-Glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines by Arthur Komlosh, Gloria Volohonsky, Noga Porat, Chen Tuby, Evgenia Bluvshtein, Pablo Steinberg, Franz Oesch and Avishay A.Stark Carcinogenesis, 22, 2009–2016, 2001 The typesetter would like to sincerely apologise for errors that appeared within this paper upon first publication in the above issue of Carcinogenesis. The correct version is republished as follows.

© Oxford University Press

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Carcinogenesis

γ-Glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines

Arthur Komlosh1, Gloria Volohonsky3, Noga Porat1, Chen Tuby1, Evgenia Bluvshtein1, Pablo Steinberg2,4, Franz Oesch2 and Avishay A.Stark1,5 1Department

of Biochemistry, Tel-Aviv University, Ramat-Aviv 69978, Israel and 2Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany

Present addresses: 3Department of Molecular Genetic, Weizmann Institute, Rehovot 76100 Israel and 4Institu¨t fur Erna¨hrungstoxikologie, Universita¨t Potsdam, Arthur Scheunert-Allee 114–116, D14558 Bergholz-Rehbru¨cke, Germany 5To

whom correspondence should be addressed Email: [email protected]

Glutathione synthesis and growth properties were studied in the γ-glutamyl transpeptidase(GGT)-negative, nontumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. γ-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/ CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with γ-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a γ-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources. Introduction γ-Glutamyl transpeptidase (GGT) is induced to high levels in many pre-neoplastic lesions (altered hepatic foci, AHF) at Abbreviations: Ab, antibody; AHF, altered hepatic foci; BSO, buthionine sulfoximine; cysteine or cystine; CG, cysteinylglycine; DMEM, Dulbecco’s modified Eagle medium; GGC, γ-glutamylcysteine; GGCS, γ-glutamylcysteine synthetase; GGCyT, γ-glutamyl cyclotransferase; GGT, γ-glutamyl transpeptidase; GS, glutathione synthetase; GSH, glutathione; HBSS, Hank’s balanced salt solution; HC, hepatocarcinogenesis; TP, transpeptidation. © Oxford University Press

early stages of hepatocarcinogenesis (HC) in rodents (1–3). The ubiquity of elevated GGT levels in many rodent and human hepatic and extrahepatic carcinomas (1–7) have led to the hypothesis that GGT provides a growth advantage to focal cells during carcinogenesis. As GGT participates in detoxification of xenobiotics, the advantage has been suggested to be due to resistance to the acute toxicity of carcinogens (1,4). However, GGT-rich AHF developed in animals treated with subacutely toxic carcinogen doses (8,9), and transfections of cells with SV40, N-ras, or rasT24 (10–12) led to increase in GGT expression and in tumorigenicity, indicating that resistance may not be the sole contributor to a growth advantage. GGT could facilitate HC by catabolism of extracellular glutathione (GSH), resulting in oxidative damage at the vicinity of AHF (13). This notion is based on the induction of oxidative damage by the GGT–GSH system in vitro (14–16), on the localization of oxidative damage in GGT-rich AHF in carcinogen-treated rats (17) and on GGT-dependent GSH mutagenesis which, in most cases, involves reactive oxygen (18–26). However, AHF may be resistant to oxidative damage (27), and antioxidants that abolish GSH–GGT-induced mutagenesis and lipid peroxidation (14,19,21) act as liver, skin or forestomach tumor promoters (28,29). Thus, the advantage may result from other GGT-dependent reactions. A plausible hypothesis is that the advantage may be due to the role of GGT in the transport of GSH constituents, leading to increase in cellular GSH. The latter is required for proliferation and resistance (3,7,30,31). At limiting cysteine concentrations, GGT cDNA-transfected mouse hepatoma cells recovered cysteine moieties and grew in vitro faster than untransfected cells, and growth depended on GGT when GSH was the exogenous cysteine source (32,33). GGT-transfectants produced tumors with an average mass 3-fold larger than vectortransfected cells (28). GSH biosynthesis is catalyzed by γ-glutamylcysteine synthetase (GGCS) and glutathione synthetase (GS). The cellular cysteine concentration and GGCS levels are ratelimiting. GGCS is feedback inhibited by GSH, leading to a steady state in cellular GSH. GGT catalyzes two reactions: hydrolysis of a γ-glutamyl bond and transpeptidation (TP). Thus, GGT may participate in promotion of growth by two mechanisms: (i) removal of a γ-glutamyl moiety from exogenous GSH by GGT and cleavage of the resulting CG by a dipeptidase provide the rate-limiting cysteine, (ii) GGTdependent TP in the presence of a γ-glutamyl donor (such as GSH or glutamine) and cyst(e)ine yields γ-glutamylcyst(e)ine (GGC) which, in turn, is readily transported (34,35), thus circumventing the rate-limiting GGCS and its feedback inhibition by GSH. The growth advantage of GGT-rich cells at limiting cysteine in a glutamine-containing medium (32) may reflect TP-dependent supply of GGC rather than supply of cyst(e)ine. As an in vitro model system for liver epithelial cells we used rat oval cell lines. Oval cells proliferate shortly after 671

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exposure to carcinogens (36), and once transformed they give rise to cholangiocellular and hepatocellular carcinomas (37–39). The oval cells OC/CDE22 are anchorage dependent, are not tumorigenic and GGT activity is not detected in cell extracts (38) or is negligible (see below). Their transformed counterparts M22 cells express GGT, are anchorage independent and induce tumors in syngeneic newborn rats (37). In this work we studied GSH biosynthesis and growth behavior of these cell lines as related to GGT activity. Materials and methods Materials and cell lines Media and supplements were from Biological Industries, Beth Haemek, Israel and Gibco-BRL, Eggenstein, Germany. Acivicin, buthionine sulfoximine (BSO) and enzymes were from Sigma, Milwaukee, WI, USA. GGC and CG disulfide was from Bachem, Torrance, CA, USA. Cell line OC/CDE22 was established from livers of rats undergoing the choline-deficient, ethioninesupplemented diet protocol at week 22 of feeding. Cell line M22 was obtained by reiterated exposures of OC/CDE-22 to the mutagenic carcinogen N-methylN⬘-nitro-N-nitrosoguanidine (37,38). Media Medium A was Dulbecco’s modified Eagle medium (DMEM): Ham’s F-10 (1:1) containing 3% newborn calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphothericin B, 2 mM L-glutamine, 1.15 µg/ml insulin, 1 µg/ml hydrocortisone and 15 mM HEPES buffer pH 7.3. This medium contains 100 mM of each cysteine and cystine (300 mM ‘cysteine equivalents’). Cysteine-deficient medium B, contained DMEM for metabolic labeling, 1% serum, supplements as above and 4.5 g/l glucose, 40 µM iinositol, 10 µM cysteine, 0.4 mM arginine, 0.8 mM leucine, 0.2 mM methionine and 0.9 mM Na2HPO4. Growth conditions Growth at limiting cysteine concentrations. Cells were seeded onto 24-well clusters (6500–8500/well) in 0.5 ml of medium A. After growth for 24 h, it was replaced by medium B containing various cysteine concentrations, and the cultures were grown up to 6 days with daily replacement of medium. Triplicate wells from each point were counted daily. Depletion of GSH. Cells (0.8–1.5⫻106/well in 6-well clusters) were grown in medium A, which was replaced by 1.5 ml medium B. Triplicate wells were assayed for GSH and protein (see below) at various time points. Repletion of GSH. Cells (0.7–2⫻106/well) were depleted of GSH for 20 h were washed in PBS and incubated in 1.5 ml Hank’s balanced salt solution (HBSS) supplemented with 30 mM HEPES buffer, pH 7.3, 12.5 mM NaHCO3, 0.5 mM pyruvate, and GSH or GSH precursors. Samples were withdrawn with time, processed and assayed for GSH and protein (see below). Processing took ⬍15 min. Significant GSH loss did not occur during this period, as judged by incubation of trypsinized cells up to 30 min at room temperature (34, 33.3 and 34.7 nmol GSH/mg protein at 9, 15 and 30 min, respectively). Antibody. Monoclonal Ab 5F10 was produced from selected hybridomas derived from mice immunized with purified native rat kidney GGT, and was purified from ascitic fluid of mice injected with hybridoma line 5F10 by ammonium sulfate precipitation. It inhibits ⬎80% of TP (at 600 µM D-γglutamyl-p-nitroanilide and 20 mM glycylglycine) but not hydrolysis (at 600 µM D-γ-glutamyl-p-nitroanilide) (40). Preparation of cell samples for the assay of GSH and protein. Cells were trypsinized, centrifuged (3000 g, 3 min), washed in phosphate-buffered saline (PBS) and resuspended in 100 ml of 10% sulfosalicylic acid and centrifuged at 14 000 g. Supernatants were immediately frozen and stored at –30°C for GSH assay. Pellets were dissolved in 1 N NaOH and were assayed for protein. GSH assay. GSH assay was as described (41), using the recycling method with GSH reductase. Replacement of NADPH by a generating system (0.33 mM NADP, 2 mM glucose-6-phosphate and 1 U/ml G6PD) yielded the same coefficient (0.252 ⫾ 0.037 A412/min for 1 µM GSH). Enzyme assays. GGT and aminopeptidase activities were determined as described (21,42) with sonicates of cells suspended in 50 mM Tris–HCl pH 8.0. GGT activity in attached whole cells was essentially as described (43), in HBSS supplemented with 30 mM HEPES buffer pH 7.3, 12.5 mM NaHCO3, 0.5 mM pyruvate, 20 mM glycylglycine and 1 mM GGPNA. GS and GGCS were assayed in the soluble protein fraction from sonicates of cells suspended in 20 mM imidazole, 0.1 mM EDTA pH 8.0 and centrifuged at 10 000 g for 50 min. In

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Fig. 1. The effect of Ab 5F10 on the growth of oval cell lines. After growth of OC/CDE22 cells (white symbols) and M22 cells (black symbols) at nonlimiting cysteine concentration (medium A) for 24 h, the medium was replaced by medium B supplemented with the indicated concentrations of cysteine, without (A) or with (B) 100 µg/ml of Ab 5F10. Cells were counted at the indicated time points. Presented are means ⫾ SD of triplicate wells.

order to prevent GSH loss, the soluble fraction was exposed to 0.2 mM acivicin for 1 h. GS was assayed as described (44), except that acidified samples (40 µl of reaction mixture in 60 µl 10% sulfosalicylic acid) were assayed for GSH as above. GGCS was assayed as described (44) except that 20 mU of purified rat kidney GS (3.5 U/mg) were added, and acidified samples were assayed for GSH as above. One unit of GS and GGCS is the amount of enzyme forming 1 µmol GSH/min. Protein was assayed with the Bradford reagent (Bio-Rad, Munchen) with bovine serum albumin as a standard. Protein contents were 281 ⫾ 50 mg/106 cells in both lines.

Results Inhibition of the transpeptidatic activity of GGT by Ab 5F10 affects growth rate GGT activity has been claimed to be responsible for the growth advantage of GGT cDNA-transfected mouse hepatoma cells at limiting cysteine concentrations (32). In order to test whether hydrolysis or TP provide this advantage, the growth of OC/CDE22 and M22 cells was followed at limiting (10 and 30 µM) or non-limiting (200 µM) concentrations of cysteine. Growth rates of both lines depended on exogenous cysteine (Figure 1A). At the same cysteine concentrations, the presence of the TP inhibitor Ab 5F10 markedly decreased the activity of GGT (Table I) and the growth rates of M22 cells, whereas those of OC/CDE22 cells were not affected (Figure 1B). The growth rates of M22 cells with Ab 5F10 were similar to those of OC/CDE22 cells without it, suggesting that TP by GGT was largely responsible for the higher growth rates of M22 cells. Both lines had comparable activities of GGCS and aminopeptidase. GSH and GS were higher in M22 cells. OC/ CDE22 cells expressed negligible amounts of GGT activity, as compared with M22 cells (Table I). The steady state of cellular GSH depends mainly on the activity level of GGCS. As both cell lines contained similar activities of this enzyme, we expected to find similar GSH levels in them. The high GSH content in GGT-rich M22 cells as compared with that in

GGT and glutathione metabolism in rat liver oval cells

Table I. Activity of enzymes involved in GSH biosynthesis and degradation in oval cell lines Cell line

OC/CDE22

M22

GSH content, nmol/mg total protein GGCS, mU/mg cytosolic protein GS, mU/µg cytosolic protein Membranes Aminopeptidase Ma GGTa GGT with acivicina GGT with Ab 5F10a Attached whole cellsc GGTd GGT with acivicind GGT with Ab 5F10d

16 ⫾ 3 (24) 0.69 ⫾ 0.31 (24) 1.66 ⫾ 0.31 (24)

34 ⫾ 5 (24) 0.84 ⫾ 0.23 (24) 4.6 ⫾ 0.44 (24)

2.3 ⫾ 0.3 (3) 0.4 (1) NDb ND

2.8 ⫾ 0.2 (3) 84 ⫾ 1 (3) ND 32 (1)

ND ND ND

75 ⫾ 14 (10) 5.1 ⫾ 0.8 (10) 30.4 ⫾ 7.1 (10)

The enzymes were assayed as described in Materials and methods. Presented are values of means ⫾ SD and (number of determinations). Pre-incubation of cells with acivicin was at 0.5 mM for 60 min. Pre-incubation with Ab 5F10 was at 100 µg/ml for 45 min. amU/mg membranal protein bNot detected. cGSH-depleted cells, as described in Material and methods, assayed in culture as described (42). dmU/mg total protein. The relatively high GGT activity in attached cells was due to the assay at 37°C (42) as compared with the assay of membranal GGT at 25°C.

GGT-deficient OC/CDE22 cells may have resulted from a GGCS-independent pathway. The high concentration of glutamine (2 mM) in the medium and GSH exported from the cells, together with cyst(e)ine could serve, respectively, as the donors and acceptor GGT substrates for TP to form GGC. Thus, TP by GGT could be responsible for the higher GSH content in M22. If GGT-mediated hydrolysis were the important activity, then feedback inhibition of GGCS by GSH, efflux of cellular GSH and its cleavage by GGT in order to reabsorb cysteine moieties for GGCS-dependent synthesis would lead to similar GSH content in both cell lines. Repletion of cellular GSH with exogenous GSH Most of cellular GSH was depleted after incubation of cultures in medium B containing 10 mM cysteine for 18 h. GSH halflife periods were 4.9 ⫾ 0.6 and 5.1 ⫾ 1.5 h for OC/CDE22 and M22, respectively. Figure 2 shows that M22 cells, but not OC/CDE22 cells utilized exogenous GSH for repletion. GSH was not repleted in cells treated with acivicin, where 93% of the GGT activity was inhibited (Table 1), indicating that GGT activity was crucial for the utilization of exogenous GSH. Blocking of membranal GGT by pretreatment with acivicin excluded the possibility of protection of the enzyme from acivicin by GSH. Repletion of GSH with GSH constituents and precursors of GSH GSH repletion was studied in GSH-depleted M22 cells that were challenged with combinations of GSH precursors. Omission of glycine, glutamate or both at non-limiting cysteine marginally affected repletion, indicating that the endogenous pool of glutamate and glycine was sufficient to support repletion. Excess of glutamate, glutamine and glycine at limiting cysteine concentrations did not support repletion (Figure 3). Thus, similar to other cell types, the cysteine concentration is rate-limiting in GSH repletion in oval cells. GGT was suggested to be responsible for the in vivo formation of GGC from GSH (donor) and cysteine (acceptor)

Fig. 2. GSH repletion with exogenous GSH. GSH was depleted from M22 (black symbols) and OC/CDE22 cells (white symbols) in medium B containing 10 µM cysteine for 18 h. Medium B was replaced at time zero with HBSS containing 200 µM GSH (s, ∆) or 200 µM GSH and 0.5 mM acivicin (j, ,). GSH and protein were determined at the indicated time points. Exposure of cells to acivicin was at 0.5 mM for 60 min prior to exposure to GSH.

Fig. 3. GSH repletion with cysteine, glutamate and glycine. GSH-depleted M22 cells were exposed at time zero to HBSS containing 20, 30 or 40 µM cysteine (Cys 20–40); 200 µM cysteine (Cys), 200 µM glycine (Gly), 200 µM glutamate (Glu) and 200 µM glutamine (Gln). At the indicated time points, sample wells were assayed for protein and for GSH.

in the kidney (34). GSH concentration in rat plasma is 26 µM (45), sufficient to fulfil the kinetic requirement of GGT (Km ⫽ 5.6 µM) (46). Although glutamine is less active than GSH as a donor (46), its abundance in plasma (close to 1 mM) (47) indicates that it could serve as a physiological γ-glutamyl donor. 673

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Fig. 4. Repletion of GSH with cystine and glutamine. GSH-depleted M22 cells (A) and OC/CDE-22 cells (B) were exposed at time zero to HBSS containing 200 µM glycine and the indicated supplements: 200 µM cysteine (Cys), 200 µM glutamate (Glu), 200 mM or 2 mM glutamine (Gln) and 100 mM cystine (Cystine). GSH and protein were determined at the indicated time points.

Cystine is the most efficient γ-glutamyl acceptor (46). We tested whether glutamine and cystine are more effective than glutamate and cysteine in GSH repletion in GGT-rich cells, and whether the transpeptidatic activity of GGT is responsible for repletion. Exposure of GSH-depleted cells to various amino acid combinations revealed that glutamine/cystine supported the highest rate and extent of repletion in M22 cells, as compared with those obtained with glutamate/cysteine. Repletion with glutamine/cystine in the presence of the TP inhibitor Ab 5F10 was similar to that obtained with glutamate/ cysteine (Figure 4). All combinations were similarly effective in GSH repletion in OC/CDE22 cells (Figure 4), indicating that TP-dependent formation of GGC may have contributed to GSH repletion in GGT-rich, but not in GGT-deficient cells. The increase of repletion with glutamine/cystine over that with glutamate/cysteine should conceivably be resistant to the GGCS inhibitor BSO. However, BSO abolished completely the repletion supported by cysteine, cystine, and CG due to GGCS inhibition, and that supported by GGC due to inhibition of the transport of γ-glutamyl peptides (35) (data not shown). It should be noted that the 33% increase of repletion in mixtures containing glutamine as compared with those containing glutamate might be less pronounced due to the relatively large standard deviations (Figure 4). The rate and extent of GGC-dependent repletion in OC/ CDE22 cells were similar to that supported by cysteine, with or without acivicin (Figure 5), indicating that GGC was transported as such into the cells and that acivicin does not inhibit this transport. The parameters of GGC-dependent repletion in M22 cells were similar to those obtained with OC/CDE22 cells exposed to GGC (Figure 5), cysteine or cystine (Figure 4), and to those obtained with M22 cells exposed to cyst(e)ine/glutamate, or cyst(e)ine/glutamine and Ab 5F10 (Figure 4). GGC-dependent repletion in M22 was inhibited by acivicin (Figure 5). These findings suggest that GGC transport in M22 may have been rendered sensitive to 674

Fig. 5. Repletion of GSH with γ-glutamylcysteine. GSH-depleted M22 cells (A) and OC/CDE22 (B) were exposed at time zero to HBSS containing, as indicated, 200 µM cysteine (d, s) or γ-glutamylcysteine (., ,) in the absence (black symbols) or the presence (open symbols) of 0.5 mM acivicin for 60 min prior to exposure. GSH and protein were determined at the indicated time points.

acivicin, and that the repletion in M22 without acivicin was due to cleavage of GGC by GGT and utilization of the resulting cysteine. Cleavage of CG is the rate-limiting step in repletion with exogenous GSH The rate and extent of GSH repletion with non-limiting exogenous GSH were approximately half of those obtained with non-limiting cyst(e)ine/glutamine or cyst(e)ine/glutamate. This was due to the requirement for cleavage of cysteine from CG: aminopeptidase M cleaves CG and is inhibited by bestatin, whereas the rat renal cysteinylglycinase cleaves CG-disulfide and is inhibited by penicillamine (42). GSH repletion with GSH or CG was half that obtained with cysteine. Inhibition of aminopeptidase M by bestatin abolished repletion with CG but not with cysteine, whereas penicillamine did not inhibit CG-dependent repletion (Figure 6). CG-disulfide by itself or with bestatin did not support GSH repletion, whereas repletion occurred with CG-disulfide and penicillamine (Figure 7). The fact that CG, but not its disulfide form, supported GSH repletion only in the absence of peptidase inhibitor indicates that cysteinylglycinase is absent from oval cells. The apparently contradictory finding that CG disulfide supported repletion in the presence of penicillamine was due to the reduction of CG-disulfide by the reactive thiol in penicillamine to form CG, which, in turn, was cleaved by aminopeptidase M. It is yet unknown whether CG enters the cells and is cleaved intracellularly, or whether cleavage occurs at the plasma membrane level. In addition to the ability of CG to replace the cysteine that is required for GSH repletion in oval cells, it has been shown to increase GSH levels in mouse kidney (33) and to promote the growth of mouse hepatoma cells (32). Discussion TP by GGT may provide an additional source for GGC that may lead to high growth rates of M22 cells The activity of the rate-limiting GGCS was similar in both lines. Thus, the higher growth rates at limiting cysteine

GGT and glutathione metabolism in rat liver oval cells

Fig. 6. Effect of inhibitors of aminopeptidase on GSH repletion, CG as the cysteine source. GSH-depleted M22 cells were exposed at time zero to HBSS containing, as indicated: 200 µM GSH, 200 µM CG (Cysgly), 10 µM bestatin, 200 µM cysteine (Cys), 400 µM penicillamine and 100 µM BSO. GSH and protein were determined at the indicated time points.

Fig. 7. Effect of inhibitors of aminopeptidases on GSH repletion, CG disulfide as the cysteine source. GSH-depleted M22 cells were exposed at time zero to HBSS containing as indicated: 200 µM GSH, 200 µM CG (Cysgly), 100 µM CG-disulfide [(Cysgly)2], 400 µM penicillamine and 10 mM bestatin. GSH and protein were determined at the indicated time points.

concentrations, the higher GSH content and the more efficient GSH repletion in M22 cells are probably to be due to the expression of GGT rather than to their higher activity of the non-rate-limiting GS. In GGT-deficient OC/CDE22 cells, the supply of GGC for GSH synthesis is solely GGCSdependent, thus repletion rates remained unchanged regardless of whether or not the GSH precursors were substrates for GGT-catalyzed TP. The extent of repletion was similar to the cellular GSH steady state in OC/CDE22 cells grown under non-limiting conditions, due to feedback inhibition of GGCS by GSH. In M22 cells, GGC is supplied by GGCS using the non-limiting glutamate and glycine, and also by GGTdependent TP, using glutamine or exogenous GSH as donors. The following support this idea. (i) The GGT-dependent portion of GSH repletion (glutamine and cystine) is GGCSindependent and is not feedback inhibited by GSH, resulting in higher rates of repletion, and reaching cellular GSH concentrations which are above the steady state governed by feedback inhibition of GGCS. Upon specific inhibition of TP by Ab 5F10, repletion rate and extent with glutamine/cystine and the cellular GSH concentrations were similar to those obtained with cysteine/glutamate, where GSH synthesis depends only on GGCS. (ii) The higher growth rates of M22 cells as compared with those of OC/CDE22 cells at limiting cysteine concentrations (Figure 5 and ref. 32) may have been due to the presence of glutamine in the growth medium, which enabled the supply of GGC via GGT. (iii) The TP-inhibitor

Ab 5F10 decreased the growth rates of M22 at limiting cysteine to those typical of OC/CDE22, suggesting that TP by GGT may promote growth by the supply of GGC, similar to the TP-dependent supply of GGC originating in GSH and cystine (48). The finding that repletion with GGC in both cell lines did not result in GSH content higher than that obtained with cysteine (Figure 5) may be explained as follows. During the synthesis of GSH from cysteine, the rate of utilization of GGC by GS exceeds that of its formation by GGCS (Table I). The above and the high affinity of GS to GGC (50 µM; ref. 49) indicate that the cellular concentration of GGC is low, especially when the latter is feedback inhibited by GSH. Under such conditions, GGC is not cleaved readily by GGCyT (Km for γ-glutamylaminobutyrate ⫽ 6.6 mM; ref. 50). The transport of exogenous GGC may, however, lead to high cellular concentrations of the latter and to efficient cleavage by GGCyT (77 U/mg protein in rat liver; ref. 51). The cellular concentration of GGC was probably low, considering the highest theoretical rate of its uptake (0.5 nmol/min; Figure 5A) and the high rate of its utilization by GS (Table I). The higher extents of GSH repletion with cystine/glutamine as compared with those obtained with cysteine/glutamate (Figure 4) also argue against the efficient cleavage of GGC by GGCyT. The plateaus in repletion with GGC (Figure 5) were not due to feedback inhibition of GGCS by GSH (Ki GSH ⫽ 2.3 mM; ref. 45) in that repletion with cysteine tapered off at much higher cellular GSH concentrations. 675

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As the uptake of amino acids and peptides depends on specific membranal transporter proteins, it is conceivable that the uptake of GGC depends on such a protein as well. This protein could be one of the known amino acids or peptide transporters, or an as yet unidentified one, hence the putative ‘GGC transporter’. This protein and GGT may act as a complex where the acceptor site of the latter is at proximity to the active site of the transporter. The newly formed GGC would be translocated directly to the transporter from the acceptor site of GGT without being released to the cytosol, resulting in efficient transport of GGC, whereas exogenous GGC may bind more readily to the GGT donor site, resulting in the cleavage of GGC. GGT has been suggested to participate in the transport of GSH and of γ-glutamyl amino acids (52–54). This may support the notion of cooperation between the ‘GGC transporter’ and GGT. Cleavage of CG as the rate-limiting step in the utilization of exogenous GSH In the cysteinylglycinase-rich rat kidney (42), exogenous CG and cysteine lead to similar rates of GSH synthesis (35). In rat liver oval cells, which lack cysteinylglycinase but contain aminopeptidase, the latter activity is rate-limiting for GSH repletion with exogenous GSH or CG. Thus, exogenous GSH per se seems a less favorable cysteine source for GSH synthesis in GGT-rich cells other than those of the proximal tubules in the kidney. The role of GSH as an efficient γ-glutamyl donor for TP in combination with cyst(e)ine in the formation of GGC remains to be evaluated. Although we obtained some evidence for the involvement of TP by GGT in the repletion of GSH, it is difficult to evaluate it accurately in the presence of GGCS activity. Inhibition of the latter by BSO leads also to the inhibition of transport of γ-glutamyl peptides. Therefore, we are developing a cell line in which the activity of GGCS can be manipulated by genetic means. What could be the reason for the prevalence of AHF expressing high GGT levels? The expression of many enzymes including those involved in GSH metabolism is altered in AHF and persists during HC (31,55). The pattern of expression of many of them is similar to that found in fetal liver (56). Various hepatomas, transformed rat embryo fibroblasts and cells transformed by erbB, src, ras and raf are low in GGCS and GS with frequent concomitant high GGT activities (57–66). This pattern indicates that proliferating fetal, preneoplastic and neoplastic cells may compensate for GGCS deficiency by increased GGT activity in order to fulfil their requirement for high cellular GSH. The appearance of fetal markers in AHF may indicate that foci originate in ‘maturation-arrested’ (67,68) or ‘ontogenetically blocked’ (69,70) stem cells undergoing differentiation, which had been affected by carcinogens. Thus high focal GGT and low GGCS may reflect the ‘maturation-arrested’ fetal-like state. Carcinogen insult to the differentiated adult hepatocytes may also lead to GGT-rich AHF. As GGT apparently provides a growth advantage in vivo, GGT-rich foci may result from selection of those initiated cells in which the expression of the GGT gene itself has been deregulated, or of those cells in which activation of oncogenes involved in mitogenic signal transduction would lead to increased GGT expression. The gradual increase in GGT levels (71) and in GSH content (72) in normal colon, primary colon carcinoma and in metastases 676

into the liver in the same individuals may be indicative of such selective processes occurring during promotion and progression. Acknowledgements We wish to acknowledge the generous donations from the Jac and Eva Feinberg Foundation, New York, from Boaz and Aliza Porat, Haifa, Israel and from Mr Freddy Furhmann and Mrs Ina Curiel, Curac¸ ao. We are grateful to Ms Rosario Heck for superb technical assistance. Supported in part by the Ella Kodesz Institute for Research on Cancer Development and Prevention, the Tel-Aviv University Cancer Biology Research Center, and by the Rekanati Foundation for Medical Research Israel. P.S. is a recipient of the Heisenberg Scholarship from the Deutsche Forschungsgemeinschaft.

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