Pierre Golstein

The Fas Death Factor

Fas ligand (FasL), a cell surface molecule belonging to the tumor necrosis factor family, binds to its receptor Fas, thus inducing apoptosis of Fas-bearing cells. Various cells express Fas, whereas FasL is expressed predominantly in activated T cells. In the immune system, Fas and FasL are involved in down-regulation of immune reactions as well as in T cell-mediated cytotoxicity. Malfunction of the Fas system causes lymphoproliferative disorders and accelerates autoimmune diseases, whereas its exacerbation may cause tissue destruction.

Molecular cloning and expression of the fas ligand, a novel member of the tumor necrosis factor family

The Fas antigen (Fas) belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family, and it mediates apoptosis. Using a soluble form of mouse Fas, prepared by fusion with human immunoglobulin Fc, Fas ligand was detected on the cell surface of a cytotoxic T cell hybridoma, PC60-d10S. A cell population that highly expresses Fas ligand was sorted using a fluorescence-activated cell sorter, and its cDNA was isolated from the sorted cells by expression cloning. The amino acid sequence indicated that Fas ligand is a type II transmembrane protein that belongs to the TNF family. The recombinant Fas ligand expressed in COS cells induced apoptosis in Fas-expressing target cells. Northern hybridization revealed that Fas ligand is expressed in activated splenocytes and thymocytes, consistent with its involvement in T cell-mediated cytotoxicity and in several nonlymphoid tissues, such as testis.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Immunoglobulin-binding factor present on and produced by thymus-processed lymphocytes (T cells)

Abstract An immunoglobulin-binding factor (IBF) has been detected in incubation supernatants from cortisone-resistant normal thymus cells and educated thymus cells but not untreated normal thymus cells. IBF is synthesized by the cells under test, since, for instance, it could not be detected in supernatants from cells incubated in the presence of inhibitors of protein synthesis. Moreover, IBF has been detected in supernatants from very small numbers of highly purified educated T cells, strongly suggesting that non-T cells are not involved in its production. IBF activity has also been found at the surface of about 1% of cortisone-resistant normal thymus cells and 4%–19% of educated thymus cells. Both cell surface and supernatant IBF have a specificity for the Fc region of immunoglobulins G. The existence of immunoglobulin-binding factors at the surface of some T cells and in supernatants from T cells (1) should be taken into consideration for the interpretation of results of experiments investigating immunoglobulin production by T cells, and (2) may well be relevant to fundamental mechanisms in T and non-T cell functions and interactions.

Apparent caspase independence of programmed cell death in Dictyostelium

During normal development, cell elimination [1,2] occurs by programmed cell death (PCD) [3], of which apoptosis [4] is the best known morphological type. Activation of cysteine proteases termed caspases [5] is required in many instances of animal PCD [6-9], but its role outside the animal kingdom is as yet unknown. PCD occurs during developmental stages in the slime mold Dictyostelium discoideum [10,11]. Under favorable conditions, Dictyostelium multiplies as a unicellular organism. Upon starvation, a pathway involving aggregation, differentiation and morphogenesis induces the formation of a multicellular fungus-like structure called a sorocarp [12], consisting mainly of spores and stalk cells, the latter being a result of cell death. Dictyostelium cell death is similar to classical apoptosis in that some cytoplasmic and chromatin condensation occurs but differs from apoptosis because it involves massive vacuolisation and, interestingly, lacks DNA fragmentation [11]. We examined whether caspase activity is required for Dictyostelium cell death. We found that caspase inhibitors did not affect cell death, although some caspase inhibitors that did not inhibit cell death impaired other stages in development and could block affinity-labelling of soluble extracts of Dictyostelium cells with an activated caspase-specific reagent. The simplest interpretation of these results is that in Dictyostelium, whether or not caspase-like molecules exist and are required for some developmental steps, caspase activation is not required for cell death itself.

Cell-death alternative model organisms: why and which?

Classical model organisms have helped greatly in our understanding of cell death but, at the same time, might have constrained it. The use of other, non-classical model organisms from all biological kingdoms could reveal undetected molecular pathways and better-defined morphological types of cell death. Here we discuss what is known and what might be learned from these alternative model systems.

Homology between reaper and the cell death domains of Fas and TNFR1.

When engaged by antibodies or by their respective li-gands, the cell surface molecules Fas and TNFR1 (the 55 kDa tumor necrosis factor [TNF] receptor) can transduce into cells a signal that often leads to cell death (Armitage, 1994; Nagata and Golstein, 1995). Fas and TNFR1 have homologous extracellular cysteine-rich domains that make them members of the nerve growth factor receptor super-family. They are also homologous in their cytoplasmic regions for a stretch of 60-70 amino acids, called the death domain, which makes them different from most other members of this superfamily. This dual but dissociated homology, the autonomous death signaling ability of the fact that they are encoded within the last exon of their respective genes (Behrmann et al., 1994) suggest that Fas and TNFR1 may derive from a chimeric ancestor molecule. Such a molecule would have brought together an extracellular region, ancestor to a nerve growth factor receptor domain, and an intracellular cell death signaling module. We reasoned that a derivative of the latter might still exist as an autonomous entity and therefore looked for an intracellular molecule involved in cell death and of about the same size as the death domains. Reaper, discovered through an elegant genetic approach by White et al. (1994), is a 65 amino acid peptide, the expression of which is both necessary and sufficient for developmental cell death and at least some types of experimental cell death in Drosophila. Mere visual inspection showed homology between reaper and the death domains of TNFR1 and, to a lesser extent, of Fas (Figure 1). This homology was particularly significant because it involves Drosophila and mammalian molecules and because all of these are able to signal cell death. Closer inspection suggested some heterogeneity in the distribution of homologies along reaper. In particular, the middle third of reaper shows the highest density of homologies with TNFRI. Reaper is involved in a pathway signaling cell death in Drosophila (White et al., 1994). Expression of reaper is both necessary and sufficient to proceed to the next step of this pathway (White et al., 1994). The emergence during evolution of a chimeric molecule covalently linking reaper to an extracellular module would have allowed direct access from the cell exterior to this cell death signaling pathway. For instance, a cell death pathway would thereby have become directly accessible via Fas to cytotoxic T cells bearing the Fas ligand (Rouvier et al., 1993; Suda …

Redundant cell death mechanisms as relics and backups.

Here we review recent observations indicating the existence of redundant cell death mechanisms. We speculate that this redundancy reflects a particular evolutionary history for cellular demise. Autophagic or apoptotic elements might have been added to a primordial death mechanism, initially improving cell dismantling and later acquiring the ability to act themselves as death effectors. The resulting redundancy of cell death mechanisms has pathophysiological implications.

Autophagy in Dictyostelium Genes and pathways, cell death and infection

The use of simple organisms to understand the molecular and cellular function of complex processes is instrumental for the rapid development of biomedical research. A remarkable example has been the discovery in S. cerevisiae of a group of proteins involved in the pathways of autophagy. Orthologues of these proteins have been identified in humans and experimental model organisms. Interestingly, some mammalian autophagy proteins do not seem to have homologues in yeast but are present in Dictyostelium, a social amoeba with two distinctive life styles, a unicellular stage in nutrient-rich conditions that differentiates upon starvation into a multicellular stage that depends on autophagy. This review focuses on the identification and annotation of the putative Dictyostelium autophagy genes and on the role of autophagy in development, cell death and infection by bacterial pathogens.

A Differential Molecular Biology Search for Genes Preferentially Expressed in Functional T Lymphocytes: The CTLA Genes

One approach to the isolation of molecules involved in T cell-mediated cytolysis stems from the postulate of a possible correlation between molecular phenotype and molecular functional involvement. Accordingly, CTL-specific molecules have been looked for, using a strategy based on the differential screening of a subtracted cDNA library. This led to the isolation and characterization of the following structures, expressed mostly (but no exclusively) in CTLs and inducible upon lymphocyte activation: CTLA-1 and CTLA-3 (serine-proteases), CTLA-4 (a member of the Ig superfamily) and CTLA-2 alpha and beta (homologues to the proregion of cysteine-proteases). The theoretical and practical limitations and the prospects of this type of approach are discussed.