Altered expression of membrane-bound and soluble CD95/Fas contributes to the resistance of fibrotic lung fibroblasts to FasL induced apoptosis
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Abstract Background An altered susceptibility of lung fibroblasts to Fas-induced apoptosis has been implicated in the pathogenesis of pulmonary fibrosis; however, the underlying mechanism is not completely understood. Here, we studied the susceptibility of lung fibroblasts, obtained from patients with (f-fibs) and without pulmonary fibrosis (n-fibs), to FasL- (CD95L/APO-1) induced apoptosis in relation to the expression and the amounts of membrane-bound and soluble Fas. We also analysed the effects of tumor necrosis factor-β on FasL-induced cell death. Methods Apoptosis was induced with recombinant human FasL, with and without prior stimulation of the fibroblasts with tumor necrosis factor-α and measured by a histone fragmentation assay and flow cytometry. The expression of Fas mRNA was determined by quantitative PCR. The expression of cell surface Fas was determined by flow cytometry, and that of soluble Fas (sFas) was determined by enzyme-linked immunosorbent assay. Results When compared to n-fibs, f-fibs were resistant to FasL-induced apoptosis, despite significantly higher levels of Fas mRNA. F-fibs showed lower expression of surface-bound Fas but higher levels of sFas. While TNF-α increased the susceptibility to FasL-induced apoptosis in n-fibs, it had no pro-apoptotic effect in f-fibs. Conclusions The data suggest that lower expression of surface Fas, but higher levels of apoptosis-inhibiting sFas, contribute to the resistance of fibroblasts in lung fibrosis against apoptosis, to increased cellularity and also to increased formation and deposition of extracellular matrix.Keywords:
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Apoptosis has been implicated in tumor development and progression. Fas (CD95) and Fas ligand (FasL) are an interacting receptor ligand pair that elicits apoptosis in many cell types. Although originally described as proteins regulating peripheral immune tolerance, accumulating evidence suggests that Fas/FasL may play an important role in carcinogenesis, tumor outgrowth, and metastasis. This review summarizes our current knowledge about the regulation of Fas and FasL expression, Fas signaling, soluble Fas production, the role(s) of Fas and FasL in hematopoietic and non-hematopoietic tumorigenesis and progression, and the potential application of Fas-induced apoptosis in cancer therapy.
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Fas-mediated apoptosis is a form of cell death that operates through a Fas-Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas-mutated lymphocytes. Irradiated RAG2–/– recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long-term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas– T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL-mediated cytotoxic effect on normal-derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas–) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas– T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment.
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The relationship between apoptosis,atresia folliculi and missed abortion mediated by Fas/FasL system
Fas/FasL were two kinds of transmembrane protein on the cell surface.The current study showed that FasL was the death factor and Fas was its receptor.When the FasL of a cell combined with the Fas of another,they could lead that the cell which expressed Fas died.Fas was named Apo-1,meaning CD95.Fas gene was located on human q23 of the chromosome 10 and the gene length was about 25 Kb.FasL gene was located on human q23 of the chromosome 1 and the gene length was about 8Kb.The Fas/FasL system was closely related to Physiological process of follicular atresia,autoimmune diseases,tumors and graft tolerance.Recently the incidence of missed abortion related to apoptosis has been attended by people and the Fas/FasL system is important in promoting apoptosis.
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Fas ligand (FasL) and its receptor (Fas, CD95, Apo-1) are a set of regulatory components in immune system. The aim of this study was to investigate the expression of Fas and FasL in the breast cancer. We were able to demonstrate that 90% of the invasive breast cancers expressed CD95L, with potential detrimental effects on the host organism. CD95 expression was lost (35%) in breast cancer, a mechanism probably involved in CD95L-mediated apoptosis resistance. INTRODUCTION Fas ligand (FasL) and its receptor (Fas, CD95, Apo-1) are a set of regulatory components of the immune system (Suda et al., 1993). Fas is a 45-kDa cell-surface receptor of the TNF/ nerve growth factor receptor family and is one of the most important death-domain receptors (Suda et al., 1993). Interaction of Fas with its ligand, FasL, induces receptor trimerization, which in turn results in the recruitment of the adapter protein FADD (Fas-associated death domain) and activation of caspases, which lead to irreversible cell damage and death (Nagata, 1995). FasL expression results in neoplastic cells becoming able to modify the immune response directed against them by affecting the tumor microenvironment in a way that favors the successful escape of the tumor from immune surveillance (Gutierrez et al., 1999). AIMS OF THE STUDY The aim of this study was to investigate the expression of apoptotic markers Fas and FasL in breast carcinomas. We were also interested in establishing a potential correlation between the expression of these two molecules and the clinical and biological parameters of the tumors. MATERIALS AND METHODS A number of 20 patients, diagnosed with mammary carcinoma and surgically treated in the III-rd Surgical Clinic of the “Sf. Spiridon” Hospital were taken into consideration. For each patient were investigated the following parameters: menopausal status, the histological type/gradding tumoral of the primary tumor and the lymph node status. The neoplastic samples were harvested in sterile medium and incubated with 0.4% collagenase, at 37C, over night. The isolated cells were centrifuged on glass slides (cytospin) and fixed in ethanol absolut for 15 min. at room temperature. The slides were incubated with a primary antibody (anti-Fas and anti-FasL respectively) followed by a secondary biotinylated antibody and the streptavidin-peroxidase complex. The reaction was developed with diaminobenzidine (DAB) and the cells were counterstained with hematoxylin. We have considered as positive those samples that expressed more than 5% Fas or FasL positive cells. In order to identify possible correlations between the Fas and FasL expression and the other parameters we have considered, the 2 test was employed and the statistical significance threshold was established at <0.05. RESULTS AND DISCUSSION Our investigation has revealed the following features: -FasL expression has been detected in 90% of the invasive breast carcinomas. -Fas and FasL co-expression has been evidenced in 65% of the cases. -Fas expression was partial lost in 35% of the cases. -We were not able to demonstrate a statistically significant association between the Fas and FasL molecules expression and the clinical and biological parameters taken into consideration (tables I and II). Table I. The expression of Fas associated with the clinical and biological parameters considered. Parameter Number of cases(n=20) Fas +Cells n% Fas –Cells n% Premenopause 4 4 (100%) 0 (0%) menopausal status Postmenopause 16 9(56,25%) 7(43,75%) CDI-G2 13 9(69,24%) 4(30,76%) CDI-G3 5 3(60%) 2(40%) histological type/gradding tumoral
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Some of the first signals that initiate programmed cell death (apoptosis) are mediated by specific receptors on the cell surface. Fas (also designated as CD95 or APO-1), which is a 45kDa protein that belongs to the tumor necrosis factor (TNF) receptor superfamily and has one transmembrane domain, has been identified as the molecule that transduces the apoptosis induction signal intracellularly (Yonehara et al., 1989; Itoh et al., 1991). Fas ligand (FasL or CD95L) is a 40-kDa protein that belongs to the TNF superfamily. Cells that express FasL interact directly with cells expressing Fas, and FasL acts as an apoptosis inducer through Fas molecules. Expression of the Fas molecule is detected in various organs, with marked expression on activated mature T and B cells and liver cells. FasL is expressed on activated mature T cells, and also in the testis. The Fas and FasL system is one of the major pathways of the induction of apoptosis, and defects of this system result in autoimmune disease, as has been shown by analyses of lpr and gld mice (Watanabe-Fukunaga et al., 1992; Takahashi et al., 1994). Swine Fas and FasL cDNAs were previously cloned, and their function has been intensively analyzed (Muneta et al., 2001). Genetic modification of Fas or FasL in swine will yield clues about the mechanism of autoimmune diseases like SLE, and aid the development of methods for the regulation of rejection of donor organs in xenotransplantation. Here we report the locations of Fas and FasL genes (designated TNFRSF6 and TNFSF6, respectively) on swine chromosomes by FISH analysis. We also confirmed these locations by radiation hybrid mapping.
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Fas and its ligand (FasL) are members of the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamilies, respectively. Fas−FasL interactions trigger controlled cell death (apoptosis) in the immune system and thus play a key role in the regulation of immune responses. Structural details of the Fas−Fas ligand interaction are currently unknown. Previously, six Fas residues were identified by mutagenesis as important for ligand binding. We have now extended our mutagenesis analysis and identified additional residues which contribute to the Fas−FasL interaction. Candidate and control residues were selected based on a molecular model of the Fas extracellular region. Although residues in all three extracellular domains were identified to contribute to binding, the Fas−FasL interaction is centered on the second TNFR-like domain. Important residues were compared to critical positions in TNFR and CD40, another member of the TNFR family.
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