Opposite Roles of FAP-1 and Dynamin in the Regulation of Fas (CD95) Translocation to the Cell Surface and Susceptibility to Fas Ligand-mediated Apoptosis
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Human melanoma is the most aggressive form of skin cancer and is extremely resistant to radiation and chemotherapy. One of the critical parameters of this resistance is down-regulation of Fas (CD95) cell-surface expression. Using TIG3 normal human fibroblasts and human melanoma cell lines, we investigated transcriptional regulation of FAP-1, a regulator of Fas translocation in the cell. Protein-tyrosine phosphatase FAP-1 (PTPN13, PTP-BAS) interacts with human Fas protein and prevents its export from the cytoplasm to the cell surface. In contrast, dynamin-2 facilitates Fas protein translocation from the Golgi apparatus via the trans-Golgi network to the cell surface. Suppression of dynamin functions by dominant negative dynamin K44A blocks Fas export, whereas the down-regulation of FAP-1 expression by specific RNA interference restores Fas export (a phenomenon that could still be down-regulated in the presence of dominant-negative dynamin). Based on the FAP-1- and dynamin-dependent regulation of Fas translocation, we have created human melanoma lines with different levels of surface expression of Fas. Treatment of these melanoma lines with soluble Fas ligand resulted in programmed cell death that was proportional to the pre-existing levels of surface Fas. Taking into consideration the well known observations that FAP-1 expression is often up-regulated in metastatic tumors, we have established a causal connection between high basal NF-kappaB transcription factor activity (which is a hallmark of many types of metastatic tumors) and NF-kappaB-dependent transcriptional regulation of FAP-1 gene expression that finally restricts Fas protein trafficking, thereby, facilitating the survival of cancer cells.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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To the best of the authors' knowledge, there have been few reports on the effect of testosterone on the apoptosis of macrophages. In this report, we studied the effect of testosterone on the apoptosis of bone marrow-derived macrophages (BMMs) and the function of the Fas/Fas ligand (FasL) system in the process. Results showed that testosterone treatment in vitro at the physiological concentration of 10 nM did not induce the apoptosis of BMMs. However, BMMs underwent apoptosis when treated at higher concentrations of testosterone (100, 200 and 400 nM). Testosterone-induced apoptosis was associated with the enhanced expression of Fas, FasL, and caspase-8. These data suggest that the Fas/FasL system may play an important role in the testosterone-induced apoptosis of macrophages.
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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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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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To explore the role of Fas gene in rhG-CSF-induced apoptosis.Mediated by lipofection (DOTAP), Fas cDNA was transferred into HL-60 cells. In situ hybridization, Western blotting and FCM analysis were used to demonstrate the successful transfection. The apoptosis percentages of HL-60 and Fas transfected HL-60 cells were compared after coculturing with rhG-CSF at the same final concentration.After cocultured with rhG-CSF (10 ng/ml) for 96 hours, the apoptosis percentage of Fas transferred HL-60 cells was significantly higher than that of HL-60 cells, and the Fas expressions could be up-regulated by rhG-CSF.Fas is an apoptosis-induction oncogene, and rhG-CSF induces apoptosis of HL-60 cells through Fas signalling pathway.
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Many chemotherapeutic drugs have been found to exert their mode of action via induction of apoptosis in cancer cells. The mechanisms involved in this process are not clear. Recent studies have shown that the Fas/Fas ligand (FasL) system is a key factor controlling apoptotic cell death. In the present study, the involvement of Fas in chemotherapeutic drug–induced apoptosis in hepatoma cell lines was investigated. Five different human hepatoma cell lines, Hep G2, Hep G2.2.15, Hep 3B, SK–Hep–1, and PLC/PRF/5, were used. It was found that they expressed different levels of Fas. However, all five cell lines were susceptible to apoptosis when treated with chemotherapeutic drugs such as 5–fluorouracil (5–FU) or cisplatin. In Hep G2 that constitutively expressed Fas, 5–FU or cisplatin treatment caused an increase in the expression of Fas before the formation of oligonucleosomal DNA fragments, a typical feature of apoptosis. However, in Hep 3B, where Fas is undetectable, apoptosis could also be induced by 5–FU or cisplatin without induction of Fas. The agonistic anti–Fas antibody (CH–11) was capable of inducing apoptosis by itself and promoted drug–induced apoptosis in Hep G2 but not in Hep 3B. The antagonistic anti–Fas antibody (ZB4) inhibited drug–induced apoptosis in Hep G2. Our results suggest that apoptosis can be induced in hepatoma cell lines via both Fas–dependent and Fas–independent pathways.
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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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