Fas and Fas ligand interactions suppress melanoma lung metastasis

Apoptosis induced by Fas (CD95) ligation is frequently lost during tumor progression; however, there is no direct evidence to support an association of Fas loss-of-function with metastatic tumor behavior. To determine whether Fas loss-of-function is critical for acquisition of the metastatic phenotype, we have compared the ability of Fas-sensitive K1735 murine melanomas to form spontaneous lung metastases in wild-type and Fas ligand-deficient mice. Fas-sensitive melanoma clones are highly tumorigenic but rarely metastatic in wild-type syngeneic mice. However, in Fas ligand-deficient mice, both the incidence and number of metastases are increased. These findings provide the first evidence that Fas-Fas ligand interactions can suppress metastasis and that tumor Fas loss-of-function may be causally linked to metastatic progression.     

Requirement of cysteine-rich repeats of the Fas receptor for binding by the Fas ligand

The Fas receptor is a member of a family of cell death receptors, including tumor necrosis factor receptor I (TNFR I), death receptor 3 and 4 (DR3 and DR4), and cytopathic avian receptor 1 (CAR1). The Fas receptor is composed of several discrete domains, including three cysteine-rich domains (CRDs), a transmembrane domain, and an intracellular domain responsible for transmitting an apoptotic signal. While the mechanism of Fas-mediated cell death has become elucidated, the requirements for Fas ligand binding to the receptor have not been fully defined. Using a series of chimeric Fc-receptor fusion proteins between the human Fas receptor and TNFR I, each cysteine-rich domain of Fas was found to be required for interaction with the Fas ligand. Interestingly, TNFR I CRD1 could partially substitute for the Fas CRD1. The importance of this domain was underscored by the analysis of a Fas extracellular mutation (C66R), which resulted in a complete loss of ligand binding. This mutation was cloned from a human patient suffering from Canale-Smith syndrome, which is characterized by autoimmunity resembling that observed in the lpr and lprcg mice. The localization of essential ligand binding domains in the Fas receptor correlated exactly with the ability of the Fas receptor fusion proteins to prevent cell death mediated by the Fas ligand.     

Fas and Fas ligand interaction is necessary for human osteoblast apoptosis

We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin-2 followed by 12-o-tetradecanoylphorbol 13-acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast-like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast-like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast-like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast-like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane-type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.     

Fas ligand is constitutively secreted by prostate cancer cells in vitro

LNCaP, DU145, and PC3 prostate carcinoma cells secrete the 27-kDa soluble Fas ligand (sFasL) into their local environment. sFasL arises from the 40-kDa membrane-bound form (mFasL), which can be found on the cell surface in the LNCaP line, as demonstrated by monoclonal antibody staining. mFasL was also found in extracts of all three cell lines, as demonstrated by Western blotting. FasL mRNA was detected not only in the cell lines, but in the normal prostate as well. sFasL protein could also be detected immunohistochemically in prostate secretions and in human semen. Cleavage of mFasL to sFasL could be inhibited by several matrix metalloprotease inhibitors without a change in the cellular levels of FasL. Prostate-derived sFasL is biologically active, as demonstrated by its induction of apoptosis in Fas-positive Ramos cells, which was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Mitoxantrone induces cellular apoptosis in all three prostate cancer cell lines. Mitoxantrone treatment and doxorubicin treatment also cause up-regulation of Fas, the cell surface receptor for FasL, in LNCaP cells, but not in DU145 or PC3 cells. Furthermore, the up-regulation of Fas expression by mitoxantrone at a high concentration was potentiated by hydrocortisone. When FasL interacts with its Fas, the Fas-bearing cell undergoes apoptosis. When LNCaP cells were treated with mitoxantrone and incubated with an anti-FasL monoclonal antibody, apoptosis was partially blocked. This not only further suggests that the sFasL is biologically active, but that the up-regulation of Fas in the presence of sFasL accounts, in part, for the cytotoxicity of mitoxantrone.     

Levels of soluble Fas ligand in myocarditis

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Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis

The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.     

Application of a Fas ligand encoding a recombinant adenovirus vector for prolongation of transgene expression

An adenovirus vector encoding murine Fas ligand (mFasL) under an inducible control was derived. In vivo ectopic expression of mFasL in murine livers induced an inflammatory cellular infiltration. Furthermore, ectopic expression of mFasL by myocytes did not allow prolonged vector-mediated transgene expression. Thus, ectopic expression of functional mFasL in vector-transduced cells does not appear to confer, by itself, an immunoprivileged site sufficient to mitigate adenovirus vector immunogenicity.     

Involvement of Fas/Fas ligand system-mediated apoptosis in the development of concanavalin A-induced hepatitis

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.     

Identification of amino acid residues important for ligand binding to Fas

OBJECTIVE: An autoantibody to a nucleolar RNA helicase protein (Gu) was recently discovered in a patient with gastric antral vascular ectasia or watermelon stomach, a disorder that is increasingly being described in systemic sclerosis (SSc). The present study was undertaken to determine whether anti-Gu antibodies occur in connective tissue diseases (CTD) and, if so, to determine their frequencies and any clinical or immunogenetic associations. METHODS: Anti-Gu antibodies were determined by Western blotting of glutathione-purified glutathione S transferase-Gu fusion proteins against consecutive antinucleolar antibody-positive sera (HEp-2 cell substrate) collected over a 5-year period in a rheumatology antinuclear antibody (ANA) testing laboratory. RESULTS: Anti-Gu antibodies were found in 11 (10%) of 108 antinucleolar antibody-positive sera. The subjects with anti-Gu antibodies included 3 of 46 patients with SSc (7%), 3 of 17 patients with systemic lupus erythematosus (18%), 4 of 9 patients with undifferentiated CTD (44%), and 1 healthy relative of an SSc patient. None of the anti-Gu-positive patients had any symptoms suggestive of watermelon stomach. Increased frequencies of both HLA-DQA1*0501 and DQB1*0301 were found, but only DQB1*0301 maintained statistical significance after correction. CONCLUSION: Anti-Gu (nucleolar RNA helicase) antibodies occur in low frequencies in patients with CTDs who have antinucleolar antibodies by ANA testing, but they are not specific for SSc or the watermelon stomach lesion.     

The Fas/Fas ligand system is involved in the pathogenesis of autoimmune myocarditis in rats

The mechanisms responsible for myocardial injury and cell death in myocarditis are still unclear. We examined whether myocardial cell death occurs via apoptosis in myosin-induced autoimmune myocarditis in rats and whether the Fas/Fas ligand (FasL) system plays a role in this apoptosis. On days 14, 17, 21, and 35 after immunization with porcine heart myosin, some cardiomyocytes and infiltrating lymphocytes were found to be apoptotic on in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay, but none was on day 60 and in control rats. Apoptotic indices peaked at day 17, and laddering of genomic DNA from the affected myocardium was observed on days 17 and 21 on agarose gel electrophoresis. The expression of Fas mRNA and protein was detected on days 17 and 21 in some cardiomyocytes and infiltrating lymphocytes by Northern blot analysis and immunohistochemistry, respectively. In addition, FasL was detected in some infiltrating lymphocytes on days 14, 17, and 21 by both in situ hybridization and immunostaining, and FasL-positive lymphocytes were mainly CD4+ cells. Some rats were injected with anti-Fas Ab (0.1 mg/kg) or anti-FasL Ab (0.1 mg/kg), and subsequently, inflammatory lesions exhibited less severe than did untreated rats with myocarditis. These findings suggest that cell death via apoptosis of cardiomyocytes and lymphocytes is one of the mechanisms of myocardial injury in autoimmune myocarditis, and that the Fas/FasL system might play a role in the induction of this apoptosis.     

Constitutive expression of Fas ligand in large granular lymphocyte leukaemia

Currently, hyperoxia is being investigated as a method for producing contrast in magnetic resonance images of the brain, solid tumors, and the eye. However, the underlying physiological mechanisms involved in this type of contrast are still not completely understood. For example, under what conditions would dissolved plasma oxygen contribute to the hyperoxia-induced contrast? Using the eye as a model system, we varied the level of dissolved plasma oxygen and observed different patterns of contrast in the vitreous. The observed contrast changes were consistent with tissue oxygen buffering by hemoglobin at an arterial PO2 of 200 mm Hg and dissolved oxygen offloading at arterial PO2's > 350 mm Hg. These data demonstrate that dissolved plasma oxygen does not become an important contrast mechanism until the arterial oxygen tension exceeds approximately 350 mm Hg. The implication of this result to studies in other organs is discussed.     

Fas/Fas ligand up-regulation and Bcl-2 down-regulation may be significant in the pathogenesis of Hashimoto's thyroiditis

Hashimoto's thyroiditis (HT) is an autoimmune disorder characterized by diffuse thyroid lymphocytic infiltration and follicle destruction. Cross-linking of the Fas receptor with its own ligand (FasL) triggers apoptosis in various systems, whereas the Bcl-2 protooncogene inhibits apoptotic cell death. The involvement of Fas, FasL, and Bcl-2 in the apoptotic process in HT was evaluated in 15 thyroid tissue samples from patients with HT stained for apoptosis and for Fas, FasL, and Bcl-2 protein expression. Eight samples from healthy thyroid tissue were used for comparison. Thyroid follicles in HT samples exhibited strong staining for Fas and FasL and a high percentage of apoptosis (30.3 +/- 14.5%, mean +/- SD), in contrast to normal control follicles that exhibited moderate Fas, minimal or no FasL, and hardly any apoptosis. Immunostaining for Bcl-2 was high in normal, and weak in involved, thyroid follicles. Infiltrating lymphocytes stained weakly for FasL and strongly for Bcl-2. We conclude that follicular cells in HT undergo apoptosis by concomitant up-regulation of FasL and Fas and down-regulation of Bcl-2 protein. The lymphocytes do not seem to be directly engaged in the process with their own FasL, but they may provide the appropriate cytokine milieu that, in turn, up-regulates Fas and/or FasL leading to apoptosis.     

Regulation of CD95 (Apo-1/Fas) ligand and receptor expression in squamous-cell carcinoma by interferon-gamma and cisplatin

CD95 (Apo-1/Fas) ligand (CD95L) expression has been observed in various malignancies. In human primary cell lines from a squamous cell carcinoma (SCC) of the vulva, the effect of cisplatin (CDDP) and IFNgamma on the expression of CD95L and its 2 receptor isoforms, CD95 transmembrane (CD95tm) and CD95 soluble receptor, was studied at the mRNA and protein levels. Addition of CDDP and IFNgamma increased CD95L mRNA levels in the primary cell line 6-fold and 1.7-fold, respectively. In comparison, CD95tm mRNA levels were diminished by CDDP but increased 8-fold upon IFNgamma challenge. CD95L expressed by SCC cells was functionally relevant since these cells were able to induce CD95-specific apoptosis in autologous lymphocytes from the SCC-bearing patient. Thus, CD95L expression in SCC may contribute to tumor-associated immunosuppression, which may be modulated by CDDP and IFNgamma. In tumor samples of the primary SCC, CD95L expression was enhanced in the area of the border between invasive tumor tissue and surrounding stroma cells. The locally restricted over-expression of CD95L was congruent with the arrangement of apoptotic stroma cells in the direct vicinity of invading tumor tongues, suggesting a role as invasion factor for CD95L.