B/C10ME Eliminates Resistance to Cell Mediated Cytotoxicity Suppression of Stress Protein GRP78 Induction in Tumor

Tumor cells undergo self-destruction when incubated with cytotoxic T-cells (CTL) consistent with the observation that suppression of target protein synthesis causes resistance to apoptosis. Resistance to CTL is also induced by stress, suggesting that pathways exist suppressing apoptosis. Here we examine whether stress induced lysis resistance to CTL and tumor necrosis factor a involves stress proteins GRP78 and GRP94. We show that inhibition of GRP78 synthesis by transfection of cells with grp78 antisense vector pRSV-78WO leads to inability to induce resistance to CTL or tumor necrosis factor a. Resistance induced in untransfected cells is reversible upon stress removal and correlates with GRP78 rephosphorylation, consistent with the notion that phosphorylated GRP78 is nonfunc tional. The possibility that GRP78 plays a role in defense against CTL mediated apoptosis is supported by the finding that CTL but not (1)4' cells express a high level of unphosphorylated GRP78. INTRODUCTION Target cell lysis by CTL2 is the in vitro manifestation for transplant rejection, a process of importance still poorly understood. Two mecha nisms causing cell death have been identified, lysis from without and lysis from within. The complement system evolved to lyse cells from without as does perforin present in CTL (1-3). Recent evidence in dicates, however, that CTL lysis proceeds via apoptosis (4, 5) not inducible by pore forming proteins. Consistent with this, target protein synthesis inhibition can interfere with CTL lysis (6). In support of this a protein that facilitates CTL mediated target lysis has recently been identified to be FAS (7). It is therefore not unexpected that cells can also activate mechanisms capable of suppressing apoptosis. Induction of stress, e.g., has been reported to induce resistance to CTL as well as the toxic effects of TNF-a (8, 9). Stress causes induction of stress proteins which increase cell survival (10-15). Although the precise protective role of stress proteins remains to be elucidated it may be caused by their ability to aid in protein folding and to regulate calcium stores in the endoplasmic reticulum (16, 17). The observation that CTL induce irreversible calcium increase in targets could provide an explanation why stress-induced targets develop resistance to CTL. Stress may, however, cause a multitude of effects, among those inhi bition of protein synthesis, which in itself causes CTL resistance (6). It therefore remains to be determined whether stress proteins are the cause of lysis resistance or the consequence of induced stress. Using various approaches we show here that lysis resistance correlates with the induction of GRP78 in a tumor target and that CTL themselves express a high level of GRP78. MATERIALS AND METHODS Animals, Cell Lines and Transfectants. C57BL/6, BALB/c and DBA/2 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The Received 6/15/93; accepted 10/7/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from the NIH to G. D. (CA37706, CA39623) and A. S. L. (R37 CA27607). 2 The abbreviations used are: CTL, cytotoxic T-cells; TNF, tumor necrosis factor; FCS, fetal calf serum; SDS, sodium dodecyl sulfate; 2-DG, 2-deoxyglucose; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; MLR, mixed lymphocyte reac tion; HSP70, heat shock protein 70. tumor target cell line B/C10ME-//-2rf was grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS (18). B/C10ME was transfected with 10 ¿¿g pRSV-78WO DNA ( 19) and 10 fig DNA of dihydrofolate reducÃ-ase plasmid pFR400 (20, 21) using the calcium phosphate precipitation method. Transformants were selected by stepwise increase of methotrexate to 5 /XMand resistant cells cloned by limiting dilution. Mixed Lymphocyte Reaction Cell Mediated Cytotoxicity Assay Induc tion of Stress, TNF-a Assay, and Lymphocyte Isolation. For induction of CTL mixed lymphocyte cultures were set up by mixing C57BL/6-//-2'" re sponder and 1000-rad irradiated BALB/c-W-2'' stimulator spleen cells in RPM1 1640 supplemented with 10% FCS, 5 X 10~5 M 2-mercaptoethanol, 2 mm glutamine, 1 HIMsodium pyruvate, and 0.1 HIMnonessential amino acids (6). On day 5, cells were harvested and used for Cytotoxicity assays. B/C10MEH-2d cells were labeled with Na251CrO4 for 60 min at 37°Cin RPMI 1640 containing 5% FCS and washed three times with Hanks' balanced salt solution containing 5% FCS. Labeled targets (104/well) were incubated in a volume of 200 fil with effector cells in RPMI 1640 in 96-well round bottomed microtiter plates. After incubation for 4 h, plates were centrifuged, and supernatants were harvested and counted in a gamma counter. The percentage of Cytotoxicity was calculated as the percentage of releasable counts after subtraction of sponta neous release (6). For induction of stress, cells were cultured in tissue culture medium con taining 5 JAMA23187, 5 ITÃŒM L-azetidine-2-carboxylic acid, 10 ITIM2-deoxyglu cose, or 1.5 /ng/ml tunicamycin all purchased from Sigma Chemical Co. (St. Louis, MO) for various lengths of time (6). To assay for sensitivity of target cells to TNF-a normal or stress induced targets (2 X IO4) were incubated for 18 h with murine recombinant TNF-a (Genzyme, Boston, MA) in the presence of 1 /xg/ml actinomycin D in a total volume of 300 /j.1of phenol red free RPMI 1640. 3-(4,5-Dimethylthiozol-3-yl)-2,5-diphenyltetrazolium bromide was added for 4 h at 37°Cand absorbance was read at 560 nm. The percentage of dead cells was calculated as the ratio of absorbance in wells with and without TNF-a (6). To isolate CD8+ cells from MLR or normal spleen, cells were passed over nylon wool columns and nonadherent cells were treated with anti-CD4 plus complement as described (18). CD4+ cells were isolated from either normal spleen passed over nylon wool columns by treatment with anti-CD8 plus complement (18) or BALB/c anti-DBA/2 MLR. Cell purity was ascertained by fluorometry on a FACStar plus (Becton Dickinson) (6, 18). Protein Labeling, Gel Electrophoresis, and RNA Hybridization. Nor mal or stress induced B/C10ME cells were harvested and labeled with 100 p.Ci of [35S]methionine for 90 min at 37°Cin methionine free medium containing 10% dialyzed fetal calf serum. After two washings in phosphate buffered saline, cells were lysed in 2X sample buffer (0.125 MTris, pH 6.8-4% SDS10% glycerol-0.02% bromophenol blue-4% ß-mercaptoethanol). Lysates with equal amounts of radioactivity were separated by electrophoresis on 10% SDS-polyacrylamide gels. Gels were stained with Coomassie blue, dried, and exposed to Kodak XAR X-ray film (6). To assay GRP78 transcript levels, aliquots were removed from cultures of normal or stress induced cells. RNA was extracted, separated on denaturing formaldehyde formamide gels by elec trophoresis, transferred to nitrocellulose sheets, and blot-hybridized with -12Plabeled hamster grp78 complementary DNA probe p315 (22). To control for RNA loading the blot was also hybridized with an actin probe and probe p3A10 (23). To determine phosphorylation of GRP78 after removal of stress, cells were labeled with 32P¡ 3 h prior to removal of stress inducer, washed, and recultured in drug free medium in the presence of 32P¡. At the time points indicated cell aliquots were harvested and cell lysates were separated by electrofocusing 2-dimensional acrylamide gel electrophoresis (10). Gels were stained with Coomassie blue and exposed to autoradiography. Intensities of Coomassie blue staining were scanned, 12P incorporation was determined and relative increases in GRP78 phosphorylation were calculated.