Oncogene (2008) 27, 1618–1628 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE The cystine/cysteine cycle: a redox cycle regulating susceptibility versus resistance to cell death A Banjac1, T Perisic1, H Sato2,6, A Seiler1, S Bannai2, N Weiss3,PKo¨ lle3, K Tschoep4, RD Issels4, PT Daniel5, M Conrad1,7 and GW Bornkamm1,7 1GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Institut fu¨r Klinische Molekularbiologie und Tumorgenetik, Mu¨nchen, Germany; 2Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 3Medizinische Poliklinik Innenstadt der Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany; 4Medizinische Klinik III der Ludwig-Maximilians-Universita¨t, GSF-Klinische Kooperationsgruppe Hyperthermie, Mu¨nchen, Germany and 5Medizinische Klinik mit Schwerpunkt Ha¨matologie und Onkologie, Charite´, Humboldt Universita¨t, Berlin, Germany The glutathione-dependent system is one of the key Introduction systems regulating cellular redox balance, and thus cell fate. Cysteine, typically present in its oxidized form cystine Redox regulation of cell cycle progression and cell death in the extracellular space, is regarded as the rate-limiting has attracted remarkable interest in recent years (Arner substrate for glutathione (GSH) synthesis. Cystine is and Holmgren, 2000). A variety of enzymatic systems transported into cells by the highly specific amino-acid are involved in the maintenance of intracellular redox À antiporter system xc . Since Burkitt’s Lymphoma (BL) homeostasis including the glutathione and thioredoxin- cells display limited uptake capacity for cystine, and are dependent systems. Starting to dissect their functional thus prone to oxidative stress-induced cell death, we stably redundancy, we have created mice with targeted À expressed the substrate-specific subunit of system xc , deficiencies for both cytosolic and mitochondrial thio- xCT, in HH514 BL cells. xCT-overexpressing cells redoxin reductases (Txnrd1 and 2, Conrad et al., 2004; became highly resistant to oxidative stress, particularly Jakupoglu et al., 2005). Thereby, we could demonstrate upon GSH depletion. Contrary to previous predictions, that Txnrd2 indeed efficiently protects cells against the the increase of intracellular cysteine did not affect the detrimental effects of GSH depletion (Conrad et al., cellular GSH pool, but concomitantly boosted extracel- 2004). lular cysteine concentrations. Even though cells were Glutathione is present in cells in millimolar concen- depleted of bulk GSH, xCT overexpression maintained tration and is considered as the major natural antioxi- cellular integrity by protecting against lipid peroxidation, dant, protecting cells from oxidative stress (Meister, a very early event in cell death progression. Our results 1995). Availability of cystine/cysteine is the rate-limiting À show that system xc protects against oxidative stress not step in GSH synthesis (Bannai and Tateishi, 1986; Ishii by elevating intracellular GSH levels, but rather creates a et al., 1987). Cysteine is transported into cells via neutral reducing extracellular environment by driving a highly amino-acid transport systems, whereas cystine, the efficient cystine/cysteine redox cycle. Our findings show predominant form in plasma, extracellular body fluids that the cystine/cysteine redox cycle by itself must be and cell culture medium, is carried by the anionic À viewed as a discrete major regulator of cell survival. amino-acid transport system, system xc (Bannai and À Oncogene (2008) 27, 1618–1628; doi:10.1038/sj.onc.1210796; Tateishi, 1986). Expression of system xc is fairly low in published online 10 September 2007 many cell types as firstly depicted for murine B lymphocytes. Provision of b-mercaptoethanol (2-ME) Keywords: cystine–glutamate exchange; glutathione me- or other sulfhydryl-containing compounds is thus a À tabolism; lipid peroxidation; redox regulation; system xc prerequisite for the survival of those cells in vitro (Broome and Jeng, 1973; Metcalf, 1976). Thiol-contain- ing compounds form mixed disulfides and thereby release cysteine which eventually enters cells via neutral amino-acid transport systems. The mixed disulfides in turn enter the cell via a transport system for bulky amino acids, releasing cysteine intracellularly. Thiol Correspondence: Dr M Conrad, Institute of Clinical Molecular Biology and Tumor Genetics, GSF-Research Centre, Marchioninistr. 25, compounds are, however, not required if B cells are co- Munich, Bavaria 81377, Germany. cultured with irradiated fibroblasts. Fibroblasts have a E-mail: [email protected] high uptake capacity for cystine, and upon intracellular 6Current address: Department of Bioresources, Faculty of Agriculture, reduction, provide cysteine to co-cultured B cells (Falk Yamagata University, Tsuruoka, Yamagata 997-8555, Japan. 7These authors contributed equally to this work. et al., 1998). Received 22 February 2007; revised 4 July 2007; accepted 20 August Limited uptake capacity for cystine is a phenomenon 2007; published online 10 September 2007 neither restricted to murine cells nor to B cells. T cells The cystine/cysteine redox cycle A Banjac et al 1619 are dependent on the supply of cysteine by antigen- transfected cells and hxCT-overexpressing cells for the presenting cells (Droge et al., 1991; Kuppner et al., time intervals of 1, 2 and 3 min. L-cystine uptake was 2003). Proliferation of many human and murine virtually undetectable in untransfected and vector- lymphoma and leukemia cell lines are dependent on transfected cells, whereas xCT-transfected cells showed free thiols or a feeder layer of irradiated fibroblasts an uptake activity of more than 2 nmol minÀ1 per mg (Falk et al., 1993, 1998). Likewise, neurons show limited protein, which was linear at least for the first 3 min. uptake capacity for cystine which may lead to low Uptake of L-cystine was efficiently inhibited in the intracellular GSH concentrations and high susceptibility presence of 2.5 mML-glutamate, indicating that L-cystine to oxidative stress-induced cell death (Murphy et al., uptake is solely mediated by the cystine–glutamate 1989, 1990). exchange transporter (Figure 1d). Seeding BL cells at À The xc cystine/glutamate-exchange transporter is a low cell density generates oxidative stress which can be heterodimer composed of the xCT light chain conferring overcome by the addition of antioxidant supplements the specificity of the amino-acid exchange reaction, and (Falk et al., 1993; Brielmeier et al., 1998). To investigate the 4F2 heavy chain, a ubiquitously expressed cell whether overexpression of human xCT provides a surface component shared with several other amino-acid growth advantage to HH514 cells, a critical cell density transport systems (Sato et al., 1999; Verrey et al., 2000). was determined that discriminates between cell survival/ The xCT light chain consists of 12 putative transmem- proliferation and cell death. Cells were plated in brane domains and is linked to the 4F2 heavy chain 96-well plates in serial dilutions from 10 000 cells per through an extracellular disulfide bond (Sato et al., well (100 000 cells mlÀ1) down to 20 cells per well. À 1999). Transcription of the xCT gene and xc transport For untransfected and vector-transfected control activities are induced by oxidative stress, mediated by cells, the critical cell density was 50 000 cells mlÀ1, electrophilic agents, depletion of cystine and by oxygen whereas overexpression of human xCT supported (Bannai et al., 1989). xCT-deficient mice are viable and cell growth to a density as little as 6000 cells mlÀ1 fertile indicating that the supply of cysteine can be (Figure 1e). compensated by other routes during normal develop- ment. By contrast, fibroblasts isolated from xCTÀ/À mice can only be cultivated if the culture medium is xCT-overexpressing HH514 cells are highly resistant to supplemented with 2-ME or N-acetylcysteine (NAC, L-buthionine-sulfoximine- (BSO) mediated cell death Sato et al., 2005). Next, we examined whether xCT-overexpression sup- À To study the contribution of system xc to the redox ports growth of BL cells even under strongly limiting balance in proliferation and prevention of human B cells GSH conditions. To this end, HH514 cells were from oxidative stress-induced cell death, we have cultivated in the presence of various L-buthionine- established a BL cell line that stably expresses xCT light sulfoximine (BSO) concentrations, and the number of chain. Our data demonstrate that elevated expression of viable and dead cells was determined over a period of 8 xCT efficiently protects BL cells from oxidative stress- days (Figures 2a and b). We used BSO in our studies, induced cell death even under conditions of cellular since BSO specifically inhibits g-glutamyl-cysteine GSH depletion. synthetase (g-GCS), the rate-limiting enzyme in GSH anabolism, and thus causes rapid depletion of intracel- lular GSH (Griffith, 1982). While control cells already died at the lowest BSO concentration within 48 h, Results proliferation of xCT-overexpressing cells was slowed down in a dose-dependent manner and cells remained L-cystine uptake is strongly increased in viable even in the presence of 100 mM BSO (Figure 2b). xCT-overexpressing cells and is sensitive to BSO treatment did not impair the uptake capacity of inhibition by L-glutamate cells for cystine (Figure 2c). Addition of 2.5 mM GSH Human and murine xCT light chain were cloned into the rescued the BSO-mediated