(CANCER RESEARCH 53. 1456-1460. March 15. 199.1] Expression of BCL-2 Protein Enhances the Survival of Mouse Fibrosarcoid Cells in Tumor Necrosis Factor-mediated Cytotoxicity1 Thierry Hennet, Giuseppe Bertoni, Christoph Richter, and Ernst Peterhans2 Institute of Veterinary- Virology, University of Berne. Länggass-Strasse 122. CH 3012 Berne ¡T.H., G. B., E. P.\: and Laboratory of Biochemistry I. Swiss Federal Institute of Technology, Zurich ¡C.R.¡,Switzerland ABSTRACT Tumor necrosis factor (TNF) kills some types of tumor cells in vitro and participates in tumor elimination in vivo. TNF has been shown to kill cells by altering their mitochondria structurally and functionally. The oncogene l<( I -2 codes for a protein located in the inner membrane of mitochondria which is able to inhibit the commitment to cell death in various cell types. We have therefore investigated whether TNF-mediated killing of the cell line L929 could be modulated by expression of the protein BCL-2. We report here that L929 cells transfected with a BCL-2 expression vector have an increased survival compared to wild type cells after TNF chal lenge. The protective effect is greatest at moderate TNF concentrations and is still significant at concentrations that killed 100% of wild type cells. The action of BCL-2 is selective inasmuch as cells are not protected against other cytotoxic agents blocking various mitochondria! functions. We show that cells expressing BCL-2 have a higher mitochondria! membrane po tential iAM'i than wild type cells. The increase in AM'could be linked with the enhanced survival of cells after TNF challenge. Indeed, we found that treatment of wild type L929 cells with the ionophore nigericin, which increases AM7,protects them even at high TNF concentrations. INTRODUCTION In human follicular B-cell lymphoma the BCL-2 gene is translo cated from chromosome 18 to chromosome 14 and its expression becomes regulated by the potent immunoglobulin heavy chain pro moters resulting in overexpression of the oncogene (1,2). The BCL-2 gene codes for a M, 26,000 membrane protein with no similarity to any other known protein (3). Its localization in the inner membrane of mitochondria suggests that it could influence some functions of this organelle (4). Beside its mitochondrial location, BCL-2 has the unique properly for an oncogene to enhance cell survival rather than to promote cell proliferation (5) as demonstrated by its protective effect against apoptotic cell death (6). However, the precise function of BCL-2 within mitochondria has not been elucidated. BCL-2 is of physiological importance in the establishment of B-cell memory (7), and its pattern of expression in different tissues suggests that it could be involved in the life span control of various cell types (8, 9). We were interested whether the expression of certain rescue pro teins can protect cells against immune effector mechanisms. This could be a way used by some tumor cells to escape immunosurveil- lance. In a first attempt, we focused our attention on the role of BCL-2 expression in tumor cells challenged by the cytotoxic cytokine TNF3 in vitro. The mechanism of TNF-mediated cell death varies according to the target cells, some dying by necrosis and others by apoptosis (10). Oxidati ve stress (11, 12), induction of endogenous nucleases (13), and depletion of ATP/NADH stores (14) have been proposed to contribute Received 9/14/92; accepted 1/5/93. The costs of publication of this article were defrayed in pan by the paymenl of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicale this fact. 1This work was supported by the Swiss National Fund, grants No 31-28810.90 to E.P., and No 31-26254.89 to C.R. 2 To whom requests for reprints should be addressed. ' The abbreviations used are: TNF, tumor necrosis factor; cDNA, complementary DNA; AM', mitochondrial membrane potential; O2 . Superoxide anión; TNF-R55, TNF receptor I (M, 55.000); TNF-R75. TNF receptor II (M, 75,000); NAO. IO-/V-nonylacridine orange; WT. wild type. to cell killing. Further, some groups have observed that cytotoxicity is related to structural and functional alterations of mitochondria in cells treated with TNF (15-17). We have shown that mitochondria of TNF- treated L929 cells generate increased amounts of O2 (18). Mitochon dria are the first cellular target to show damage, and the loss of mitochondrial integrity leads to cell death apparently because of a depletion of energy stores. Since the BCL-2 protein is located within mitochondria, we inves tigated whether it can protect TNF-treated cells by preventing mito chondrial damage. To this end, we concentrated on the mouse fibro- sarcoid L929 cell, one of the most thoroughly studied targets of TNF cytotoxicity. We report that expression of BCL-2 in L929 cells en hances the resistance of these cells to TNF-mediated cytotoxicity. Protection is specific inasmuch as cells are still sensitive to an array of other cytotoxic agents. In addition, we show that the effect of BCL-2 is not due to a change in the expression of TNF receptors or to a decreased ability to produce O2 after TNF addition. Since BCL-2 is located in mitochondria, we also investigated the mitochon drial electron transport system in BCL-2-transfected cells. While the activity of the mitochondrial respiratory chain remains unchanged, we observed a higher Aty in cells expressing BCL-2. This parameter could be linked to the TNF resistance inasmuch as we found that the ionophore nigericin, known to increase A^, protects the cells against killing by TNF. MATERIALS AND METHODS Materials. Recombinant mouse TNFa was purchased from Genzyme (Boston, MA). The activity was 4 X IO7 units/mg protein. The mouse BCL-2 cDNA was a generous gift of S. J. Korsmeyer (Howard Hughes Medical Institute, St. Louis, MO). W. Lesslauer (Hoffmann LaRoche Ltd., Basel, Switzerland) kindly provided the cDNA of mouse TNF receptors. The expres sion vector BCMGSNeo was obtained from F. Melchers (Basel Institute for Immunology, Basel, Switzerland) (19). Unless indicated the chemicals were from Sigma Chemical Co. (St. Louis, MO). Cell Culture. The mouse fibrosarcoid L929 cell line was obtained from the American Type Culture Collection (Rockville, MD). The cells were subcloned by means of a cloning ring and the derived subline (WT) was used for all further experiments. The cells were cultured in Dulbecco's modified Eagle's medium (Giòco, Paisley, United Kingdom), supplemented with 2 mw glutamine, 10 mm 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (pH 7.4), penicillin (100 units/ml), streptomycin (100 ug/ml), and 7% heat- inactivated fetal calf serum. Plasmid Construct. All enzymes and buffers were from Boehringer Mann heim (Mannheim, Germany). The BCL-2 gene was excised from the pN2-M- Bcl-2 vector with tfmdIII and EcoRl (5) and ligated into the polylinker of the Bluescript plasmid at the WmdIII and EcoRI sites (Stratagene, La Jolla, CA). The resulting construct was then digested with Xhol and Noti and the 926-base pair BCL-2 fragment was inserted into the corresponding sites of the BCMG SNeo vector. Electroporation and Selection of G-418-resistant Cells. Cells were har vested after trypsinization, washed twice with ice cold phosphate-buffered saline and resuspended at 1 x IO7 cells/ml. Plasmid DNA (25 ug) was added to the cells which were electroporated after 10 min incubation on ice using a Gene Pulser apparatus (Bio-Rad, Richmond, CA) set at 1000 V, 25 uF. Cells were then cultured in Dulbecco's modified Eagle's medium-7% fetal calf serum. The antibiotic G-418 (Gibco, Madison, WI) was added at 0.5 mg/ml to the culture medium after 48 h. Batches of 10,000 cells were transferred to 1456 on May 3, 2021. © 1993 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from