[CANCER RESEARCH54, 4851—4854, September 15, 1994] Advances in Brief Bcl-2 Inhibits T-Cell-mediated Cytolysis of a Leukemia Cell Line1 Toshihiko Torigoe, Juan A. Milan, Shinichi Takayama, Russell Taichman,2 Toshiyuki Miyashita, and John C. Reed3'4 Oncogene & Tumor Suppressor Gene Program, La Jolla Cancer Research Foundation, La Jolla. California 92037 cells against antibody-based responses, since it does not prevent complement-mediated cell death (10) and does not prevent phagocy tosis (1 1), as might occur, for example, when tumor cells are coated with antibody. The potential impact that Bcl-2 might have on cell mediated immune responses, however, is more difficult to predict because of the multiple mechanisms that CTLs, NK cells, LAK cells, and tumor infiltrating lymphocytes have at their disposal for inducing target cell death (reviewed in Ref. 12) and because the predominant mechanism used appears to vary among different populations or clones of T-, NK, LAK, and tumor-infiltrating lymphocyte cells. In this regard, cell-mediated cytolysis can occur through mechanisms consistent with either apoptosis or necrosis, depending on the partic ular immune cell effectors and the target cells studied (reviewed in Ref. 13). For the most part, C'TLs, NK, and related cells kill tumor targets through two separate processes: (a) secretion of cytotoxic granules which contain a number of potentially lethal molecules including perform (a protein that resembles the C9 component of complement and that pokes holes in membranes), AlT (which can stimulate apopotosis in some types of cells probably via purinergic receptors), proteases (that trigger DNA fragmentation and apoptosis through a pathway which may require cdk kinases), and TIA-1 (an RNA-binding protein that induces apoptosis through poorly under stood mechanisms); and (b) expression of genes encoding cytotoxic cytokines, such as TNFc-a, lymphotoxins, and the Fas ligand (14— 18). With regards to the latter, gene transfer-mediated elevations in Bcl-2 protein levels have been shown to provide at least partial protection against apoptotic cell death induced by TNF-a and anti bodies to Fas (19, 20), suggesting that Bcl-2 could potentially render tumor cells more resistant to CTL-induced killing in scenarios where local secretion of TNF-a or expression of transmembrane forms of TNF-a or Fas ligand play a predominant role in the cytolytic mech anism. On the other hand, if perform-dependent mechanisms involv ing loss of osmotic equilibrium and necrotic cell death predominated, Bcl-2 would not be expected to provide resistance, by analogy to the lack of protection seen previously for complement-mediated lysis (10). In fact, it has been reported by others that Bcl-2 does not protect against cytolysis induced by at least some allospecific CFLs (10, 21). We show here, for the first time, however, that gene transfer-mediated elevations in Bcl-2 protein levels can render a human leukemia cell line relatively more resistant to DNA fragmentation and cytolysis induced by a cloned T-cell that induces cell death through mecha nisms consistent with apoptosis. Abstract The bcl-2 gene becomes dysregulatedin its expressionin a wide variety of human cancers and has been shown to block both spontaneous and drug induced cell death, thus conferring a selective survival advantage on malig smut cells. The biochemical mechanism by which bcl-2 promotes cell survival remains enigmatic but appears to involve a downstream event in an evolu tionarily conserved cell death pathway. Here we report that gene transfer mediated increases in Bd-2 protein levels in the human leukemia line Jurkat render these cells more resistant to induction of DNA fragmentation and cytolysis by a cloned T-celL The kiffing mechanism used by these particular T-cells was consistent with apoptosis, as opposed to necrosis, in that DNA degradation occurred as a prelysis event. The findings raise the possibffity that dysregulation ofbcl-2 gene expression could play a role in the avoidance of immune surveillancemechanisms by cancer cells. Introduction The bcl-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations that frequently occur in non-Hodgkin's B-cell lymphomas and which move the bcl-2 gene from its normal location at 18q21 into a cis-configuration with strong enhancer elements associated with the immunoglobulin heavy-chain locus at 14q32 (1). High levels of Bcl-2 protein production have also been reported in a wide variety of human solid tumors and leukemias in the absence of translocations or other gross alterations in the structure of the bcl-2 gene, including adenocarcinomas of the prostate and colon, small cell and non-small cell carcinomas of the lung, nasopharyngeal carcinomas, neuroblastomas, acute myelogenous leu kemias, and chronic lymphocytic leukemias (for examples, see Refs. 2—6).The Bcl-2 protein has been shown to contribute to neoplastic cell expansion by blocking the normal physiological turnover of cells that occurs due to programmed cell death (reviewed in Ref. 7). In addition, gene transfer-mediated elevations in Bcl-2 protein levels have been shown to render leukemia and tumor cells relatively more resistant to induction of apoptosis by multiple types of chemothera peutic drugs (8, 9). Bcl-2, therefore, may play a significant role, not only in the origins of cancer, but also in its treatment. Yet another aspect of tumor biology that determines the potential for individual neoplastic clones to gain a selective growth advantage in vivo concerns the issue of the immune surveillance mechanisms that help to eradicate or keep in check abnormal cells in the body. In this regard, both humoral (antibody-mediated) and cellular responses can play a role in host defenses against cancer. Overproduction of the Bcl-2 protein would probably not be expected to protect malignant Received 6/20/94; accepted 8/1/94. The cost 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. I This study was supported in part by American Cancer Society Grant IM-708 and NIH Grant CA-54957. 2 Present address: Department of Periodontics, School of Dentistry, University of Michigan, Ann Arbor, MI 48109. 3 To whom requests for reprints should be addressed, at Oncogene & Tumor Suppres tar Gene Program, La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037. 4 Scholar of the Leukemia Society of America. Cells and Cell Culture. The cytolytic properties, immunophenotype, and maintenance in culture of the particular clone of IL-2-dependent CFLL-2 cytolytic T-cells and various transfectants thereof have been described in detail previously (22). CTLL-N-LCK and CT'LL-A-LCK cells represent transfectants that received a G418-resistant plasmid in combination with expression plas mids producing either normal p56-Lck or an “activated― version of Lck that contains a tyrosine—'phenylalanine substitution at position 505. Jurkat-NEO and Jurkat-BCL-2 cells are polyclonal populations of 0418- resistant Jurkat T-cell leukemia cells that were subjected to electroporation 4851 Materials and Methods Research. on October 4, 2021. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from