Cell Death and Differentiation (1999) 6, 1174 ± 1181 ã 1999 Stockton Press All rights reserved 13509047/99 $15.00 http://www.stockton-press.co.uk/cdd Review Fas ligand, death gene MJ Pinkoski*,1 and DR Green*,1 or actinomycin D. Rather than protecting against cell death, both of these agents induced significant death in HL-60 cells, 1 Division of Cellular Immunology, La Jolla Institute for Allergy and thus demonstrating that not all apoptosis was dependent on Immunology, 10355 Science Center Drive, San Diego, California, CA 92121, transcription and translation. In fact, this provided an inkling USA of the complexity of programmed cell death that would soon * Corresponding authors: MJ Pinkoski and DR Green, Division of Cellular become evident. However, the fact remained that in some Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, California, CA 92121, USA. Tel: (858) 558-3500; cases, transcription is required for apoptosis to occur and it is Fax: (858) 558-3525; E-mail: [email protected]; [email protected] now pretty much agreed that the requirement for novel RNA and protein synthesis in apoptosis is dependent on the cell type and the apoptotic stimulus. In those cases when Received 2.8.99; revised 1.10.99; accepted 5.10.99 apoptosis is dependent on transcription, the genes that Edited by L Fesus must be transcribed have been termed `death genes'. So- called death genes are those that are not transcriptionally active unless there is some sort of death stimulus. Without Abstract proper activation of the appropriate death gene(s), the The concept of death genes goes back to the early days of ultimate goal of cell death cannot be achieved in some programmed cell death, when a researcher's model system settings. Here, we will discuss one set of scenarios where this is the case. was required to be dependent on transcription of the dying cell in order to qualify as apoptosis. In 1987 Andrew Wyllie,1 one of the pioneers of cell death research, outlined four `cardinal Fas ligand as a T cell death gene elements' of apoptosis: one of which was a requirement for The cytotoxic protein, FasL, that binds the surface molecule macromolecular synthesis. In the following years the Fas was identified in 1993 as a type II membrane protein of complexity of the apoptotic process has become evident 40 kDa that is expressed on lymphoid cells as well as in and while it is now clear that apoptosis does not have to rely on tissues involved in peripheral deletion and sites of immune gene expression,the idea of death genes remains. Induction of privilege.8 FasL has also been isolated in a soluble form from an apoptotic cascade via activation of caspases, selective patients with large granular lymphocytic (LGL) leukemia and 9 release of mitochondrial proteins and further activation of natural killer (NK) cell lymphoma. FasL is a member of the caspases, can be stimulatedby engagement of the Fas surface tumor necrosis factor (TNF) family of proteins and induces apoptosis in cells bearing Fas (CD95/APO-1) on the plasma molecule via membrane bound or soluble forms of Fas ligand membrane by setting in motion a sequential activation of what (FasL). The FasL gene, which is often transcriptionally is becoming a well-defined process. inactive, becomes activated in many forms of transcription/ FasL engages surface Fas, a member of the TNF translation dependent apoptosis. Here we will discuss FasL as receptor family.10 Trimerization of Fas results in recruitment a candidate death gene. of the death inducing signaling complex, DISC, which includes the accessory molecules FADD and pro-caspase- Keywords: apoptosis; CD95 ligand; immune homeostasis and 8 as principal components (see11,12). Activation of caspase- privilege; gene regulation 8, in turn, activates the execution phase of the death program, which appears to follow one of two routes: via Introduction direct cleavage and activation of caspase-3 or by indirectly causing the release of mitochondrial cytochrome c. Upon Much of the early characterization of apoptosis was release, cytochrome c is recruited to the apoptosome with performed in thymocytes treated with either antibody to APAF-1 and pro-caspase 9 which leads to nuclear and engage the T cell receptor (TCR) or by treatment with cellular degradation. Although more complex than outlined glucocorticoid.2±6 In these situations, thymocyte death is here, the two pathways can be described according to the dependent on RNA and protein synthesis because apoptosis involvement of proteins recruited to the DISC and potential was not observed when the cells were treated in the presence inhibition by bcl-2 family members. (A more comprehensive of actinomycin D, an inhibitor of RNA synthesis, or in the review of the caspase family of proteases and the initiation presence of cycloheximide, an inhibitor of protein synthesis. and execution phases of apoptosis, which are beyond the However, it was later shown that not all apoptosis requires scope of this article, can be found in references 13±15). new RNA or protein synthesis by the condemned cell. In 1990, Martin et al.7 reported that the human promyelocytic cell line, HL-60, treated with the calcium ionophore, A23187, AICD proceeds via FasL upregulation or the microtubule disruptors, colchicine and vinblastine was One of the most compelling arguments implicating FasL as a not rescued from apoptosis by the presence of cycloheximide death gene is its involvement in activation induced cell death Fas ligand, death gene MJ Pinkoski and DR Green 1175 (AICD). After clonal expansion of lymphocytes in response to mice arose from spontaneous mutations that manifested as antigen, the immune system is faced with the task of clearing severe lymphoproliferative disorders (see Cohen and Eisen- vast numbers of activated lymphocytes. Failure to do so would berg24). In 1992, the mutation responsible for the lpr result in T cells running amok, posing the potential risk of self- (lymphoproliferation) phenotype was identified in the Fas destructive activity. Apoptosis was shown to be the mode of gene.25 Similarly, a mutation in the human Fas gene has been AICD responsible for peripheral deletion of activated described and, as in the lpr mouse, is associated with lymphocytes, that is, ones that have been primed and are lymphoproliferative and autoimmune disorders.26 In 1994, immunologically reactive16 (see Figure 1). Takahashi et al. identified the gld defect as a point mutation in Shi et al.17 reported that AICD of thymocytes and T cell the FasL gene that results in FasL mRNA being expressed but hybridomas was inhibited by cyclosporin A and the same the protein rendered nonfunctional.27 Both of lpr and gld mice year Ucker et al.18 demonstrated that AICD required de have massive accumulation of activated T cells, perhaps due novo RNA and protein synthesis, suggesting the involve- to an inability to clear peripheral lymphocytes as a result of ment of death genes. The death gene involved in AICD inoperative Fas-FasL mediated apoptosis in these cells. was later identified as FasL by four separate research groups early in 1995 who showed that FasL expressed in response to TCR stimulation mediated its apoptotic effect Stress induced FasL expression in T cells through interaction with Fas displayed on the surface of Recently another physiological system in which transcrip- activated T cells.19±22 Although TNF can also induce tion-dependent apoptosis occurs via the upregulation of apoptosis in activated T cells,23 FasL-mediated AICD is FasL has received considerable attention. Stress, in the believed to be a primary mechanism responsible for form of heat shock, genotoxic insult or radiation, has been limiting the number of effector T cells following immune demonstrated to occur via apoptotic death that can be responsive expansion. associated with activation of the FasL gene and be at least partially dependent on the presence of functional FasL protein. Killing by DNA damaging agents, long the Lessons from the lpr colony, or, there's gld in them mainstay of antitumor chemotherapy, has been shown to thar mice involve Fas/FasL for efficient killing in some cases.28 ± 30 Perhaps the most useful and informative tools in the study of When CEM and Jurkat T cells were treated with the Fas and FasL are the lpr and gld mutant mice. Each of these chemotherapeutic agent, doxorubicin, these cells under- went apoptosis which was associated with activation of the FasL gene.28 As with AICD, the apoptosis observed in these cells is characterized by the increased expression of Fas-L mRNA, which is required for death. Fulda et al.31 demonstrated this phenomenon with doxorubicin, cis-platin and VP16, all of which have DNA damage as a primary mode of action. Kasibhatla et al.30 outlined a mechanism for FasL expression induced by DNA damage in activated lymphocytes which is discussed below. The involvement of the Fas-FasL trigger to induce apoptosis in these systems is supported by the observation that g-radiation and heat shock treatment of splenic cells from lpr mice did not produce as profound an apoptotic response as from wild- type lymphocytes.29 FasL in negative selection: does too, does not, does too AICD in the thymus manifests as negative selection, the process by which self-reactive thymocytes are deleted before leaving the thymus. In the original papers on autonomous killing of activated T cells through Fas-L,19 ± 22 the observation was made that this phenomenon provided a possible explanation for death of thymocytes during negative selec- tion, a proposal that was made previously based on the differential expression of Fas during T cell ontogeny.32 However, studies with lpr mutant, FADD knockout (FADD7/7) and FADD dominant negative transgenic mice Figure 1 Schematic outline of cells expressing FasL.