Induction of Cystine Transport Activity by Stress TETSURO ISHII, HIDEYO SATO, KENJI MIURA, JUN-ICHI SAGARA, AND SHIRO BANNAI Department of Biochemistry Institute of Basic Medical Sciences University of Tsukuba Tsukuba 305, Japan Glutathione (GSH) protects cells against a wide range of insulting agents. W e have shown in cultured fibroblasts, macrophages, and vascular endothelial cells that the rate-limiting precursor for GSH synthesis is cystine in the culture medium and that the GSH level is regulated by the transport of eystine. We have been studying an Na+-independent, anionic amino acid transport system highly specific for cystine and glutamate. This system, designated as System x;, is the major transport system for cystine. Cystine transport activity in cultured human fibroblasts is enhanced in response to diethyl maleatc trcatment.’ Diethyl maleate bears an electrophilic center and reacts with GSH. Cystine transport activity is also induced in cells by sodium arsenite, cadmium chloride, and glucose/glucose oxidase which generates hydrogen peroxide.2 Induction of cystine transport activity causes an increase in GSH content of the cells. Therefore, it constitutes a protective mechanism related to thc stress caused by these agents. Oxygen concentration is also an important factor in the induction of cystine transport activity,3 because cysteine is easily oxidized to cystine by molecular oxygen in the extracellular fluid. The ambient oxygen concentration of 20% normally used in cell cultures is higher than that to which cells are exposed in vivo. The cultured cells tend to be exposed to oxidative stress more severely than are the cells in vivo. Under these conditions expression of cystine transport activity appears necessary to maintain intracellular pools of cysteine and GSH enough to survive or to grow. Actually 1. most of cells maintained in vitro have System x, activity, as summarized in TABLE

TABLE1. System x; Activity in Cultured Cell Lines or Strains Cells

Origin

IMR-90 HeLa NC-37 WI-26Vaa

Human lung fibroblast Human cervix carcinoma Human lymphoblast Human lung fibroblast, SV-40 transformed Chinese hamster ovary Porcine kidney epithelium Rat hepatoma Rat glioma Mouse lymphoma

CHO LLC-PKI HTC Ch L1210-I

System x; Activitya (pmol/min/mg protein)

Remarks

850 3 20 190 910

Bannai & Kitamura5

310 160 750 550 140

Issels et al. Foreman et al.’ Makowske & Christensen8 Hishinuma rt a/.‘)

aSodium-independent, glutamate (2.5 mM)-inhibitable uptake of cystine (0.05 mM). 497

ANNALS NEW YORK ACADEMY OF SCIENCES

498

TABLE 2. System x; Activity in Isolated Cells Svstem x; Activitv" Cells

Immediately after After 12-24 Hours Isolation in Culture

Remarks

+++

Human rnonocytes Human neutrophils Human lymphocytes Human umbilical vein endothelial cells Rat fetal brain Rat hepatocytes Mouse peritoneally exudated cellsb (1 day after stimulation, granulocytes) (4 days after stimulation, macrophages) Mouse spleen lymphocytes

-

+++

++ +

Takada & Bannai'"

ND"

++ -

Watanabe & Bannail' Ishii et al.12

OSodiurn-independent, glutamate-inhibitable cystine uptake (pmol/min/mg protein); 500. hStimulated with thioglycollate broth. 'Not determined because of cell death.

+

++

TABLE2 summarizes the cystine transport activity in isolated and primary cultured cells. So far examined, rat fetal brain cells and mouse peritoneally exudated granulocytes have the activity of System xL: when they were isolated. The activity of System x; was not found in isolated monocytes and endothelial cells, hepatocytes, and macrophages, but was soon induced upon culture. Compared with these cells, lymphocytes and neutrophils are defective in System x; activity even in culture in vim. Therefore, lymphocytes in vitro depend on cysteine which can be provided either by other cells like monocytes or by 2-mercaptoethanol, a carrier of cysteine. The protein component(s) for cystine transport may fall into the category of the stress protein, although it is not yet identified. We are trying to isolate a cDNA clone encoding the cystine transporter using an oocyte expression ~ y s t e m . ~ REFERENCES 1. 2. 3. 4. 5. 6.

7. 8.

9. 10.

11. 12.

BANNAI, S. 1984. J. Biol. Chern. 259 2435-2440. S., H. SATO,T. ISHII& S. TAKETANI. 1991. Biochim. Biophys. Acta 1092: 175-179. BANNAI, S., H. SATO,T. ISHII& Y. SUGITA. 1989. J. Biol. Chem. 264: 18480-18484. BANNAI, ISHII, T., K. NAKAYAMA, H. SATO,K.MILIRA, M. YAMADA, K. YAMADA, Y. SUGITA& S. BANNAI. 1991. Arch. Biochem. Biophys. 289 71-75. BANNAI, S. & E. KITAMURA. 1980. J. Biol. Chem. 2 5 5 2372-2376. ISSELS, R. D., A. NAGELE,K. ECKERT & W. WILMANNS. 1988. Biochem. Pharrnacol. 37: 881-888. J. W., J. LEE& S. SEGAL.1988. Biochem. Biophys. Acta 968 323-330. FOREMAN, 1982. J. Biol. Chem. 257: 5663-5670. MAKOWSKE, M. & H. N. CHRISTENSEN. I., T. ISHII,H. WATANABE & S. BANNAI. 1986. In Vitro22: 127-134. HISHINUMA, 1984. J. Biol. Chem. 259 2441-2445. TAKADA, A. & S. BANNAI. H. & S. BANNAI. 1987. J. Exp. Med. 165: 628440. WATANABE, & S. BANNAI. 1987. J. Cell. Physiol. 133 330-336. ISHII,T., Y. SUGITA