APOPTOSIS IN BURKITT LYMPHOMA CELLS IS PREVENTED BY PROMOTION OF CYSTEINE UPTAKE Martin H. FALK 1,3 , Thomas MEIER 2 , Rolf D. ISSELS 2 , Markus BRIELMEIER 1 , Beatrix SCHEFFER 1 and Georg W. BORNKAMM 1 * 1 Institute of Clinical Molecular Biology and Tumour Genetics, GSF–National Research Center for Environment and Health, Munich, Germany 2 Institute of Clinical Haematology, GSF–National Research Center for Environment and Health, Munich, Germany 3 Medizinische Klinik III, Klinikum Grosshadern, Ludwig-Maximilians University, Munich, Germany Burkitt lymphoma (BL) cells are highly sensitive to sub- optimal growth conditions and undergo apoptosis when seeded at reduced serum concentration or low cell density. Irradiated fibroblasts can protect BL cells from apoptosis induced by lowering the serum concentration or cell density through secretion of a survival- and proliferation-promoting activity which is soluble and labile. Murine B cells have a restricted uptake capacity for cystine and require cysteine for proliferation, which can be supplied efficiently by feeder cells. Therefore, we have studied the role of cysteine and other compounds with free thiol groups for survival and prolifera- tion of BL cells. Cysteine, when added alone, exerted strong toxicity on BL cells. This toxicity could be counteracted by the addition of catalase, pyruvate or bathocuproine disulfo- nate (BCS), all of which interfere with the production of hydrogen peroxide. Inhibition of the toxicity of cysteine was necessary to unravel the survival- and growth-promoting activity of cyst eine at low cell density. -Thioglycerol, -mercaptoethanol and dithiothreitol had similar toxic activ- ity in the absence of catalase, pyruvate and BCS and, through stimulation of cysteine uptake and glutathione synthesis, displayed a similar survival- and growth-promoting activity in the presence of the protective agents. The survival- and proliferation-inducing activity of thiol compounds in the presence of catalase, pyruvate and BCS was not associated with induction of BCL-2 or BAX . Cysteine/cystine uptake and the intra/cellular glutathione level are thusimportant param- eters, determining the susceptibility vs. resistance of BL cells to apoptosis. Int. J. Cancer 75:620–625, 1998.  1998 Wiley-Liss, Inc. Burkitt’s lymphoma (BL) is a malignant human B-cell tumor occurring with a high incidence in tropical areas of Central Africa and New Guinea. These cases show a high association (95%) with the Epstein-Barr virus (EBV). BL occurs with a lower incidence and a lower EBV association (20%) all over the world (for review, see Lenoir and Bornkamm, 1987). Establishing cell lines from BL cases from low-incidence areas, Lenoir et al. (1985) noted that the use of a feeder layer of irradiated human fibroblasts greatly improved the frequency of outgrowth of cell lines. Most of these cell lines became feeder-independent upon prolonged in vitro cultivation, whereas some cell lines retained a significant feeder dependence when cultured at sub-optimal conditions, i.e., at low cell density or low FCS concentration (Falk et al., 1993). With- drawal of the feeder cells induced apoptosis under these conditions. Modulating the susceptibility of BL cells to apoptosis by changing the culture conditions has become an important model system to study the role of EBV and viral gene products in protection from apoptosis (Gregory et al., 1991; Falk et al., 1993; Henderson et al., 1993) and induction of BCL-2 (Henderson et al., 1993; Rowe et al., 1994). In view of the importance of this model system, we were surprised to note that no information is available about the mechanism by which the susceptibility of BL cells to apoptosis is modulated by the culture conditions. We were inter- ested to gain mechanistic insight into this question. Attempts to characterize the apoptosis-preventing and growth- stimulating activity provided by irradiated fibroblasts at a molecu- lar level have been unsuccessful so far. Conditioned media of irradiated fibroblasts as well as a large number of defined cytokines were not able to protect BL cells from apoptosis induced by their withdrawal from the feeder layer (Falk et al., 1993). Retinol, described as a growth factor for human B cells (Buck et al., 1990), as well as -mercaptoethanol (-ME), a growth factor for murine B cells (Ishii et al., 1981a), were inactive in our system. However, the fibroblast-mediated growth stimulation turned out not to require cell-to-cell contact (data not shown), which implied a soluble and labile nature of the fibroblast activity. Furthermore, the activity provided to murine B cells by irradiated fibroblasts is also soluble and labile (Ishii et al., 1981b). Since in this system cysteine plays a key role in mediating growth- and survival-promoting effects, we studied whether cysteine exerts effects on BL cells. During these experiments, we noted a marked toxicity of cysteine (Issels et al., 1985; Nath and Salahudeen, 1993) against BL cells, which could be counteracted by the addition of catalase, pyruvate or bathocuproine disulfonate (BCS). Inhibition of the toxic activity of cysteine was necessary to unravel the strong survival- and growth-promoting activity of cysteine for BL cells. Uptake of cysteine/cystine is the limiting factor in the maintenance of a reducing intra-cellular environment and, thus, governs the cell’s susceptibility to apoptosis induced by low FCS and low cell density. Our data highlight the importance of the intra-cellular anti-oxidant defense system in the regulation of apoptosis in human B cells. MATERIAL AND METHODS Cell lines and chemicals BL41 and BL70 (EBV-negative), BL29 (EBV-positive) and LCL IARC171 (Lenoir et al., 1985) were provided by Dr. G. Lenoir (Lyon, France); the EBV-positive lines CHEP-BL, ELI-BL, MWI- BL, MAK-BL and MUTU-BL (Rowe et al., 1987) by Dr. A. Rickinson (Birmingham, UK); HH514, a sub-clone of the P3HR-1 line, by Dr. G. Miller (New Haven, CT); and Karpas422, a cell line derived from a follicular lymphoma (Dyer et al., 1990), by Dr. A. Karpas (Cambridge, UK). Cells were routinely grown in RPMI 1640 containing 10% FCS (PBS-Orgenics, Illkirch, France) supple- mented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1 μg/ml amphotericin B and 2 mM glutamine. BL70 cells were routinely kept in 5% FCS on a feeder layer consisting of confluent human fibroblasts (MRC5 strain; Flow, Meckenheim, Germany) irradiated with 50 Gy 2 to 10 days prior to usage (Falk et al., 1993). All other chemicals were from Sigma (Deisenhofen, Germany) or GIBCO (Eggenstein, Germany). Assays for proliferation and apoptosis BL cells were seeded at varying cell densities and varying FCS concentrations (see ‘‘Results’’) in RPMI 1640 (supplemented as above) in flat-bottomed, 96-well plates (Falcon, Becton Dickinson, Heidelberg, Germany) in triplicate. Standard deviation was usually Contract grant sponsors: Deutsche Forschungsgemeinschaft; Fonds der Chemischen Industrie. *Correspondence to: Institute of Clinical Molecular Biology and Tumour Genetics, Haematologikum der GSF, Marchioninistr. 25, D-81377 Munich, Germany. Fax: 49-89-70 99 500. Received 2 August 1997; Revised 4 September 1997 Int. J. Cancer: 75, 620–625 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer