Early nucleolar disorganization in Dictyostelium cell death.

Cell death occurs in all eukaryotes, but it is still not known whether some core steps of the cell death process are conserved. We investigated this using the protist Dictyostelium. The dissection of events in Dictyostelium vacuolar developmental cell death was facilitated by the sequential requirement for two distinct exogenous signals. An initial exogenous signal (starvation and cAMP) recruited some cells into clumps. Only within these clumps did subsequent cell death events take place. Contrary to our expectations, already this initial signal provoked nucleolar disorganization and irreversible inhibition of rRNA and DNA synthesis, reflecting marked cell dysfunction. The initial signal also primed clumped cells to respond to a second exogenous signal (differentiation-inducing factor-1 or c-di-GMP), which led to vacuolization and synthesis of cellulose encasings. Thus, the latter prominent hallmarks of developmental cell death were induced separately from initial cell dysfunction. We propose that (1) in Dictyostelium vacuolization and cellulose encasings are late, organism-specific, hallmarks, and (2) on the basis of our observations in this protist and of similar previous observations in some cases of mammalian cell death, early inhibition of rRNA synthesis and nucleolar disorganization may be conserved in some eukaryotes to usher in developmental cell death.

Hypotonic cell swelling stimulates permeability to cAMP in a rat colonic cell line.

This study characterized the membrane permeability to cAMP in a cell line derived from the rat colon (CC531(mdr+)) by comparison of fluxes of 3H-cAMP, 3H-8-bromo-cAMP, 3H-taurine, 3H-adenosine and 3H-5'AMP under various experimental conditions including cell membrane depolarization and hypotonic cell swelling. Cell volume was modified by changing the osmolality and composition of the extracellular medium. Incubation in iso- and hypotonic KCl media induced graded increases in cell volume and stable activation of volume-sensitive channels that was reflected in an increased efflux of 3H-taurine. Incubation in hypotonic KCl solution also enhanced the efflux of 3H-8-Br-cAMP (a non-hydrolysable analogue of cAMP). Both the efflux of 3H-taurine and of 3H-8-Br-cAMP were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 100 microM) suggesting the involvement of volume-sensitive anion channels. To gain further insight into the route mediating cAMP permeability, the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine were determined over short (5-min) periods. Uptakes of these substrates demonstrated close similarities: comparable increases were observed that correlated with the increases in cell volume in iso- and hypoosmotic KCl media; they were inhibited strongly by NPPB (100 microM) and metabolic inhibitors (deoxyglucose, 20 mM together with the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 10 microM) while barely reduced by dipyridamole (100 microM) and they were not affected by adenosine (1 mM). In contrast, the uptakes of 3H-adenosine and 3H-5'AMP had strikingly different properties; they were insensitive to cell swelling; barely inhibited by NPPB (100 microM) and metabolic inhibitors (deoxyglucose and FCCP) while strongly reduced by dipyridamole (100 micro M). Unlike the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine, the uptakes of 3H-adenosine and 3H-5'AMP were reduced in Na(+)-free media, suggesting the presence in this cell line of two different adenosine carriers, one sodium-dependent and one sodium-independent. Taken together the present data show that in this rat colonic cell line, cAMP permeability is increased by cell swelling in hypotonic KCl medium and inhibited by NPPB and metabolic inhibitors. The similarity of these characteristics to those of taurine permeability suggests the involvement of a volume-sensitive anion pathway.

Inhibition of basolateral cAMP permeability in the toad urinary bladder.

1. The effect of sulphonylurea drugs on hydrosmotic flow across toad urinary bladder epithelium was re-evaluated in the present study. Glibenclamide, added to the basolateral medium, significantly enhanced the osmotic flow induced by low doses of antidiuretic hormone (ADH) or forskolin (FK), while it inhibited the effect of exogenous cyclic adenosine monophosphate (cAMP) or its non-hydrolysable bromo derivative, 8-Br-cAMP, added to the basolateral medium. These opposite effects of glibenclamide on the transepithelial osmotic flow can be explained by a reduction of cAMP permeability across the basolateral membrane of the epithelium. The decrease in cAMP permeability leads, according to the direction of the cAMP gradient, to firstly an enhanced osmotic flow when cAMP is generated intracellularly by addition of ADH and FK, glibenclamide reducing cAMP exit from the cell, and secondly a decreased osmotic flow in response to cAMP (and 8-Br-cAMP) added to the basolateral medium, glibenclamide inhibiting, in this case, their entry into the cell. 2. The demonstration that glibenclamide actually inhibits the basolateral cAMP permeability rests on the fact that firstly it decreases the release of cAMP into the basolateral medium by about 40 %, at each concentration of ADH or forskolin tested, secondly it increases the cAMP content of paired hemibladders incubated in the presence of ADH or FK, when intracellular degradation was prevented by phosphodiesterase inhibition, and thirdly it decreases also the uptake of basolateral 8-Br-[3H]cAMP into paired toad hemibladders. 3. Taken together, the present data demonstrate that glibenclamide inhibits the toad urinary bladder basolateral membrane permeability to cAMP, most probably by a direct interaction with a membrane protein not yet indentified but distinct from the sulphonylurea receptor.

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.

P-glycoprotein inhibition by glibenclamide and related compounds.

Glibenclamide is well known to interact with the sulphonylurea receptor (SUR) and has been shown more recently to inhibit the cystic fibrosis transmembrane conductance regulator protein (CFTR), both proteins that are members of the ABC [adenosine 5'-triphosphate (ATP)-binding cassette] transporters. The effect of glibenclamide and two synthetic sulphonylcyanoguanidine derivatives (dubbed BM-208 and BM-223) was examined on P-glycoprotein, the major ABC transporter responsible for multidrug resistance (MDR) in cancer cells. To this end, we employed different cell lines that do or do not express P-glycoprotein, as confirmed by Western blotting: first, a tumour cell line (VBL600) selected from a human T-cell line (CEM) derived from an acute leukaemia; second, an epithelial cell line derived from a rat colonic adenocarcinoma (CC531(mdr+)) and finally, a non tumour epithelial cell line derived from the proximal tubule of the opossum kidney (OK). Glibenclamide and the two related derivatives inhibited P-glycoprotein because firstly, they acutely increased [3H]colchicine accumulation in P-glycoprotein-expressing cell lines only; secondly BM-223 reversed the MDR phenomenon, quite similarly to verapamil, by enhancing the cytotoxicity of colchicine, taxol and vinblastine and thirdly, BM-208 and BM-223 blocked the photoaffinity-labelling of P-glycoprotein by [3H]azidopine. Furthermore, glibenclamide is itself a substrate for P-glycoprotein, since the cellular accumulation of [3H]glibenclamide was low and substantially increased by addition of P-glycoprotein substrates (e. g., vinblastine and cyclosporine) only in the P-glycoprotein-expressing cell lines. We conclude that glibenclamide and two sulphonylcyanoguanidine derivatives inhibit P-glycoprotein and that sulphonylurea drugs would appear to be general inhibitors of ABC transporters, suggesting an interaction with some conserved motif.

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.

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.

[How to follow treated and untreated chronic hepatitis B and C]. / Comment suivre les hépatites chroniques virales B et C traitées et non traitées.

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.

Expression of CFTR in human and bovine thyroid epithelium.

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the thyroid has not been documented to date, although a role for CFTR in the thyroid follicular epithelium is suggested both clinically, by the occurrence of subclinical hypothyroidism in patients with cystic fibrosis (CF), and physiologically, by the presence of low-conductance, adenosine 3',5'-cyclic monophosphate-activated Cl channels in the follicular cells. Using reverse transcriptase-polymerase chain reaction with nested primers derived from exons 13 and 14 of the human CF gene, we have now documented the presence of CFTR mRNA in the human thyroid. Western blot analyses using six antibodies directed against different domains of human CFTR showed that a 165-kDa band was present in membrane extracts from bovine and human thyroid. This protein has the predicted size of mature CFTR and was not detected with preimmune serum or preadsorbed antiserum. By immunofluorescence and immunoperoxidase, CFTR was located in the follicular cells, with a diffuse, intracellular labeling pattern. Quantitative analysis revealed that 64% of the follicles were CFTR positive, but only 16% of the follicular cells were stained per follicle. The number of CFTR-positive cells was inversely proportional to the size of the follicle. These results 1) demonstrate the expression of CFTR at the mRNA and protein levels in human and bovine thyroid follicular cells and 2) suggest that CFTR expression could be instrumental in follicular enlargement.

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.

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.

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.

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.