Biomedical subjects
P Golstein
Publications and source records attributed to P Golstein.
Methods to study cell death in Dictyostelium discoideum.
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Signal transduction. FasL binds preassembled Fas.
The binding of a ligand to its receptor has always been viewed as the trigger for signal transduction to ensue. However, as Golstein explains in his Perspective, new findings (Chan et al. and Siegel et al.) suggest that the Fas receptor preassembles into trimers without the help of its ligand, and that this preassembly conditions ligand binding, and thus subsequent signal transduction of a death signal.
Interdigital cell death can occur through a necrotic and caspase-independent pathway.
Programmed cell death in animals is usually associated with apoptotic morphology and requires caspase activation. Necrosis and caspase-independent cell death have been reported, but mostly in experimental conditions that lead some to question their existence it in vivo. Loss of interdigital cells in the mouse embryo, a paradigm of cell death during development [1], is known to include an apoptotic [2] and caspase-dependent [3] [4] mechanism. Here, we report that, when caspase activity was inhibited using drugs or when apoptosis was prevented genetically (using Hammertoe mutant mice, or mice homozygous for a mutation in the gene encoding APAF-1, a caspase-activating adaptor protein), interdigital cell death still occurred. This cell death was negative for the terminal-deoxynucleotidyl-mediated dUTP nick end-labelling (TUNEL) assay and there was no overall cell condensation. At the electron microscopy level, peculiar 'mottled' chromatin alterations and marked mitochondrial and membrane lesions, suggestive of classical necrotic cell death, were observed with no detectable phagocytosis and no local inflammatory response. Thus, in this developmental context, although caspase activity confers cell death with an apoptotic morphotype, in the absence of caspase activity an underlying mechanism independent of known caspases can also confer cell death, but with a necrotic morphotype. This cell death can go undetected when using apoptosis-specific methodology, and cannot be blocked by agents that act on caspases.
WW domain-containing FBP-30 is regulated by p53.
A subtractive cloning approach was used to clone genes transcriptionally induced in thymocytes undergoing programmed cell death after gamma-irradiation. We thus identified about 60 upregulated genes. One of these genes encodes a WW domain previously briefly reported by others, which defines this gene as FBP-30. cDNA sequencing showed FBP-30 to be remarkably conserved in mammals. FBP-30 expression, essentially restricted to T cells, was regulated by p53 since it increased (1) after gamma-irradiation in wild-type but not in p53-/- thymocytes and (2) in cells transfected with a conditional temperature-sensitive p53 mutant, less than 1 h after shifting to permissive temperature. Upregulation of FBP-30 expression thus depends upon p53 protein expression and correlates with cell death induction in these systems. While the kinetics of its induction are rapid, the observed increased expression of FBP-30 in the presence of protein synthesis inhibitors suggests that FBP-30 gene expression is indirectly regulated by p53, through downregulation of a labile inhibitor of FBP-30 expression.
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.
An insertional mutagenesis approach to Dictyostelium cell death.
Programmed cell death (PCD) in Dictyostelium shows a pattern of ordered degeneration similar to that observed in higher eukaryotes but somewhat different from the most studied form of PCD, i.e. apoptosis. To contribute to a genetic definition of this process, Dictyostelium HMX44A cells have been subjected to insertional mutagenesis, followed by selection based on several rounds of differentiation/regrowth to recover only cells resistant to death. We describe here the approach used, a partial characterization of the first mutant thus obtained called C5 showing some dissociation of cell death signs, and, in this case where plasmid rescue was not possible, as a first step towards identification of the gene at play recovery of genomic flanking sequences via genomic recircularization and PCR. This work demonstrates the feasibility of an insertional mutagenesis approach to obtain death-resistant mutants in Dictyostelium.
Dismantling in cell death: molecular mechanisms and relationship to caspase activation.
The notion of a cell death programme was introduced in view of the reproducibility of its occurrence in time and space (e.g. in the developing embryo) and of its genetic determination. Programmed cell death can be schematically subdivided into three steps: a signalling phase, an execution phase and a dismantling phase. This review focuses on the latter. Apoptosis is the most studied form of dismantling of animal cells. The molecular pathways leading to certain apoptotic lesions appear to be dependent on the proteolytic activity of caspases. Death itself can, however, be caspase-independent. Also, non-apoptotic forms of cell death exist, even in animal cells; their molecular bases are still unknown. The relationship between cell death, apoptosis and caspases is discussed.
Cell death: TRAIL and its receptors.
A cell-death signaling system has been described recently that involves the ligand TRAIL and corresponding TRAIL-specific cell-surface receptors. These include two receptors able to transduce a death signal and, as a previously unsuspected control mechanism, two other receptors able to prevent this transduction.
Fas and other cell death signaling pathways.
The Fas system ensures, within the immune system, one of the two main pathways of T-cell mediated cytotoxicity, and, importantly, at least part of the downregulation of immune responses. Recently, Fas has been increasingly implicated in other functions, such as protection of immune privileged tissues and disposal of cells undergoing genomic alterations. The Fas system can be viewed as a cell death signal, linking extracellular information to the cell death execution stage. In this review, the Fas pathway will be described, compared to the other known T-cell mediated cytotoxicity mechanism, and then more generally to other cell death signals. The general features of cell death signaling will be emphasized as well as the peculiarities of the Fas system.
[How to follow treated and untreated chronic hepatitis B and C].
Viral hepatitis B and C are important causes of chronic liver disease, cirrhosis and hepatocellular carcinoma. Indications for the treatment of these two forms of chronic viral hepatitis are outlined as well as the practical modalities for the follow-up.
Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists.
Human immunodeficiency syndrome (HIV) infection leads to a progressive loss of T-cell-mediated immunity associated with T-cell apoptosis. We report here that CD4+ and CD8+ T cells from HIV-1-infected persons are sensitive to Fas (CD95/APO-1)-mediated death induced either by an agonistic anti-Fas antibody or by the physiologic soluble Fas ligand, although showing no sensitivity to tumor necrosis factor alpha-induced death. CD4+ and CD8+ T-cell apoptosis induced by Fas ligation was enhanced by inhibitors of protein synthesis and was prevented either by a soluble Fas receptor decoy or an antagonistic anti-Fas antibody. Fas-mediated apoptosis could also be prevented in a CD4+ or CD8+ T-cell-type manner (1) by several protease antagonists, suggesting the involvement of the interleukin-1beta (IL-1beta)-converting enzyme (ICE)-related cysteine protease in CD4+ T-cell death and of both a CPP32-related cysteine protease and a calpain protease in CD8+ T-cell death; and (2) by three cytokines, IL-2, IL-12, and IL-10, that exerted their effects through a mechanism that required de novo protein synthesis. Finally, T-cell receptor (TCR)-induced apoptosis of CD4+ T cells from HIV-infected persons involved a Fas-mediated death process, whereas TCR stimulation of CD8+ T cells led to a different Fas-independent death process. These findings suggest that Fas-mediated T-cell death is involved in acquired immunodeficiency syndrome (AIDS) pathogenesis and that modulation of Fas-mediated signaling may represent a target for new therapeutic strategies aimed at the prevention of CD4+ T-cell death in AIDS.
T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines.
Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.
Lymphocyte activation: T-cell regulation by CTLA-4.
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The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity.
The requirement for target cell nuclei in the two apoptotic death pathways used by cytotoxic lymphocytes was tested using model effector systems in which the granzyme and Fas pathways of target damage are isolated. Mast cell tumors expressing granzymes A and B in addition to cytolysin/perforin lysed tumor target cells about 10-fold more efficiently than comparable effector cells without granzymes. Enucleated cytoplast targets derived from these cells were also lysed with a similar 10-fold effect of granzymes. In contrast to cytoplasts, effector granzyme expression did not influence lysis of red cell targets. The Fas pathway was assessed using the selected cytotoxic T lymphocyte hybridoma subline d11S, which lysed target cells expressing Fas but not those lacking Fas. Similarly, cytoplasts derived from Fas+ but not Fas- cells were also readily lysed by these effector cells. Thus, neither the nucleus itself nor the characteristic apoptotic nuclear damage associated with the two major cell death pathways used by cytotoxic lymphocytes are required for cell death per se.
Homology between reaper and the cell death domains of Fas and TNFR1.
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