Induction Of Fas-mediated Apoptosis Research Articles

Induction of Fas-Mediated Apoptosis by Interferon-γ is Dependent on Granulosa Cell Differentiation and Follicular Maturation in the Rat Ovary.

ABSTRACTFas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

Anti-TOSO antibody treatment promotes T cell activation-induced cell death (AICD) in vitro and in vivo

T cell activation-induced cell death (AICD), that involves the induction of Fas-mediated apoptosis, is very important for the maintenance of immune homeostasis. TOSO was firstly described as an inhibitor of Fas-mediated apoptosis and overexpressed in chronic lymphocytic leukemia. Recently, TOSO was identified as IgM FcR. In this study, we produced anti-TOSO monoclonal antibody (mAb) that could block the binding of IgM to TOSO and found that T cell apoptosis is negatively correlated with TOSO expression during T cell activation. Treatment of activated T cells with anti-TOSO blocking mAb promoted T cell AICD in in vitro AICD model, and treatment of xenogeneic-GVHD mice with the antibody also increased the sensitivity of activated T cells to Fas-induced apoptosis, which was accompanied by reduction of c-FLIPL expression and up-regulation of AP-1 complex. In summary, our data indicate the anti-apoptotic effect of TOSO in T cell AICD and open up new therapeutic prospects for the treatment of hematologic malignancies and immune disorders.

Intraarticular gene delivery of CTLA4-FasL suppresses experimental arthritis

T lymphocytes are key inflammatory cells contributing significantly to the pathogenesis of Rheumatoid arthritis (RA). Biological treatments targeting T lymphocytes may provide an efficient approach for treatment of RA. CTLA4-FasL, a fusion product of extracellular domains of CTLA4 and FasL, integrating two inhibitory elements against T cells into one molecule, might be a desirable derivative of engineered soluble FasL or CTLA4 and have therapeutic potential in RA. The aim of this study was to investigate whether simultaneous induction of Fas-mediated apoptosis and blockade of co-stimulation signal by CTLA4-FasL gene delivery has a suppressive effect on adjuvant-induced arthritis (AIA) in Lewis rats. Recombinant adeno-associated virus (rAAV) vectors encoding rat CTLA4-FasL fusion gene (rAAV.CTLA4-FasL) or enhanced green fluorescent protein (rAAV.EGFP) were injected intraarticularly into both ankle joints after immunization. The ankles were monitored by measures of clinical, histological and inflammatory cytokines' changes. Treatment using rAAV.CTLA4-FasL resulted in a significant suppression of AIA compared with rAAV.EGFP control, as reflected in the mainly clinical signs including articular index, ankle joint thickness and paw swelling and typically histological characters of arthritic joints including synovial hyperplasia, inflammatory cells infiltration and cartilage degradation. Treatment with rAAV.CTLA4-FasL also significantly decreased the levels of key proinflammatory cytokines in AIA joints. Moreover, local productions of transgene mRNA and protein of CTLA4-FasL were found in injected joints without systemic distribution. Our results indicate that rAAV.CTLA4-FasL profoundly suppressed experimental model of RA, implicating the potential therapeutic applications for suppression of RA by local joint delivery of CTLA4-FasL.

Changes of Cyclins/CDKs in Fas-mediated apoptosis and the mechansim

Objective To study the changes of Cyclins/CDKs expression in Fas-mediated apoptosis, and the action mechanism in leukaemia cell lines and peripheral blood lymphocytes (PBL) in vitro.Methods The target cells--leukaemia cell lines and activated PBL stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, and the changes of Cyclins expression that related to the duration of inducing apoptosis was detected by Cyclin/DNA bi-parameter flow cytometry. Rb protein and the phosphorylation site of CDK1 and CDK2 were checked by Western blotting. Results Cyclin D3 expression was increased and Cyclin E expression decreased evidently, and the expression of Cyclins A and B1 had no significant change before the induction of Fas-mediated apoptosis in Jurkat, Molt-4 cell lines and PBL which entering cell cycle progression, till the late stage of inducing apoptosis in these cells the expression of Cyclin D3 was decreased, while Cyclin E increased. Meanwhile, apoptosis couldn' t be induced by rhFas ligand in G0-phase PBL. The level of CDK2 Thr-160 phosphorylation was decreased through the induction of Fas-mediated apoptosis evidently. The phosphorylation of CDK1 had no significant changes. Conclusion Fas-mediated apoptosis was located at late G1-phase and determined by whether the target cells had passed through the restriction point to cell division cycle or not. And the cell cycle specificity of Fas-mediated apoptosis was the result of the cells with DNA damage being blocked at late G1-phase,due to the decrease of Cyclin E expression and hypophosphorylation of CDK2 and under the surveillance of G1-phase check point. The changes of Cyclins expression at the late stage of Fas-mediated apoptosis is the result of cell arrest at G1-stage. Key words: Apoptosis; Flow cytometry

High Frequency of 1p36.32 Deletion or Loss of Heterozygosity in Follicular Lymphoma (FL).

Background: The initial genetic event in ∼85% of follicular lymphomas (FL), the most common B-cell lymphoma in North America, is the t(14;18)(q32;q21) resulting in over-expression of the anti-apoptotic protein Bcl-2. The secondary events associated with disease progression are not well understood. Alterations affecting the p arm of chromosome 1 are evident by standard karyotype analysis in ∼20% of FL. We have further examined the relationship between 1p deletion and FL using high resolution genomic analyses.Methods: The prevalence of 1p alterations was investigated in 139 cases of indolent and transformed FL using whole genome tiling path BAC array Comparative Genomic Hybridization (array CGH). Array-based single nucleotide polymorphism analysis was performed on a subset of cases using Affymetrix 500K SNP arrays.Results: Array CGH identified a minimum region of deletion spanning ∼0.5MB within 1p36.32 in 51 cases (37%). In 38 cases (27%) this loss was exhibited in the transformed sample but not the pre-transformation sample. The majority of cases displayed heterozygous deletion, while two cases showed homozygous deletion. The mechanisms of loss included simple deletions, unbalanced translocations with various partner chromosomes and eleven cases with an unbalanced t(1;1)(p36;q12). The Affymetrix 500 SNP array analyses showed copy neutral loss of heterozygosity or acquired uniparental disomy (aUPD) in three of ten cases that were negative for loss by aCGH. Contained within the 1p36.32 minimally deleted region are only a few candidate genes including tumor necrosis factor receptor superfamily 14 (TNFRS14), which has been implicated in growth inhibition of HT-29 human colon adenocarcinoma cells and induction of Fas-mediated apoptosis in non-Hodgkin's lymphoma.Conclusions: Our data indicate that loss of heterozygosity at 1p36.32 through deletion or aUPD constitutes the most common secondary cytogenetic event in FL. LOH at 1p36 may represent an important step in the progression of indolent to transformed FL. Further studies have been initiated to investigate other possible gene inactivation events such as methylation and mutation.

Epidermal growth factor receptor inhibitors enhance susceptibility to Fas-mediated apoptosis in oral squamous cell carcinoma cells

Molecular inhibition of epidermal growth factor receptor (EGFR) signaling is a promising cancer treatment strategy. We examined whether inhibition of EGFR signaling would affect the susceptibility of oral squamous cell carcinoma (OSCC) cells to Fas-mediated apoptosis. Treatment of OSCC cells with an anti-EGFR monoclonal antibody, C225, and an EGFR tyrosine kinase inhibitor, AG1478, which target the extracellular and intracellular domains of the receptor, respectively, inhibited phosphorylation of EGFR and its downstream effector molecule Akt and amplified the induction of Fas-mediated apoptosis. In OSCC cells treated with EGFR inhibitors, Fas-mediated apoptosis was accompanied by caspase-8 activation but not Bid cleavage. Caspase-3 and -8 inhibitors reduced the effect of EGFR inhibitors on Fas-mediated apoptosis in OSCC cells, but a caspase-9 inhibitor did not. These results indicate that the pro-apoptotic activity of EGFR inhibitors in OSCC cells depends on the extrinsic pathway of the caspase cascade. Although EGFR inhibitors did not affect the expression of Fas, the Fas-associated death domain protein, or procaspase-8 in OSCC cells, the inhibition downregulated cellular FLICE-inhibitory protein (c-FLIP). Moreover, knockdown of c-FLIP in HSC-2 cells with a small interfering RNA strongly enhanced Fas-mediated apoptosis. These results suggest that the EGFR signaling pathway may, in part, regulate Fas-mediated apoptosis in OSCC cells through c-FLIP expression.

Cell cycle dependency of caspase activation in Fas‐induced apoptosis in leukemia cells

The relationship between apoptosis and the cell cycle remains unclear. In the present study we have investigated the relationship between cell cycle progression and the activation of caspases (caspase-3 and caspase-8) in Fas (CD95)-mediated apoptosis in asynchronously growing leukemia cells. We found that cells expressing the active form of caspase-3 were cyclin A/B1 and Ki-67 negative but cyclin E positive, whereas expression of the active form of caspase-8 was detected in cyclin A/B1/E-negative and Ki-67-negative cells. In addition, both the activation of caspases and Fas-mediated apoptosis were completely abolished when leukemia cells were arrested in early G1 phase. Using post-sorting western blot analysis, we demonstrated that caspase-3 and caspase-8 were activated in p27-negative cells. These results suggest that caspase-3 would be activated in cells entering into late G1 or early S phase, and caspase-8 would be activated in middle or late G1 phase. The speed of cell cycle progression from G1 to S phase might be influential in the speed of caspase activation and induction of Fas-mediated apoptosis.

Differential induction of nuclear factor-kappaB and activator protein-1 activity after CD40 ligation is associated with primary human hepatocyte apoptosis or intrahepatic endothelial cell proliferation.

CD40, a tumor necrosis factor receptor superfamily member, is up-regulated on intraheptatic endothelial cells (IHEC) and epithelial cells during inflammatory liver disease, and there is evidence that the functional outcome of CD40 ligation differs between cell types. Ligation of CD40 on cholangiocytes or hepatocytes results in induction of Fas-mediated apoptosis, whereas ligation of IHEC CD40 leads to enhanced chemokine secretion and adhesion molecule expression. We now report that differential activation of two transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), in primary human hepatocytes or IHEC, is associated with and may explain, in part, the different responses of these cell types to CD40 ligation. CD40 ligation induced a rise in NF-kappaB activity in hepatocytes,which peaked at 2 h and returned to baseline by 24 h; however, IHEC CD40 ligation resulted in a sustained up-regulation of NF-kappaB (>24 h). In hepatocytes, CD40 ligation led to sustained up-regulation of AP-1 activity >24 h associated with increased protein levels of RelA (p65), c-Jun, and c-Fos, whereas no induction of AP-1 activity was observed in IHECs. Analysis of mitogen-activated protein kinase phosphorylation (phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase 1/2) and expression of inhibitor kappaBalpha were entirely consistent, and thus confirmed the profiles of NF-kappaB and AP-1 signaling and the effects of the selective inhibitors assessed using electrophoretic mobility shift assay or Western immunoblotting. CD40 ligation resulted in induction of apoptosis in hepatocytes after 24 h, but on IHECs, CD40 ligation resulted in proliferation. Inhibition of (CD40-mediated) NF-kappaB activation prevented IHEC proliferation and led to induction of apoptosis. Selective extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase inhibitors reduced levels of apoptosis in (CD40-stimulated) hepatocytes by approximately 50%. We conclude that differential activation of these two transcription factors in response to CD40 ligation is associated with differences in cell fate. Transient activation of NF-kappaB and sustained AP-1 activation is associated with apoptosis in hepatocytes, whereas prolonged NF-kappaB activation and a lack of AP-1 activation in IHECs result in proliferation.

A unique combination of inflammatory cytokines enhances apoptosis of thyroid follicular cells and transforms nondestructive to destructive thyroiditis in experimental autoimmune thyroiditis.

Treatment of cultured primary human thyroid cells with IFN-gamma and TNF-alpha uniquely allows the induction of Fas-mediated apoptosis. To investigate the role of this cytokine combination in vivo, CBA/J mice were immunized with thyroglobulin and then injected with IFN-gamma and TNF-alpha. Compared with control animals, mice treated with IFN-gamma and TNF-alpha showed significantly sustained lymphocytic infiltration in the thyroid, which was associated with the destruction of portions of the follicular architecture at wk 6 after initial immunization. Furthermore, the number of apoptotic thyroid follicular cells was increased only in the thyroids from mice treated with the IFN-gamma and TNF-alpha. We also analyzed the function of the Fas pathway in vivo in cytokine-treated mice by using an agonist anti-Fas Ab injected directly into the thyroid. Minimal apoptosis of thyroid epithelial cells was observed unless the mice were pretreated with IFN-gamma and TNF-alpha. These data demonstrate that this unique combination of inflammatory cytokines facilitates the apoptotic destruction of thyroid follicular cells in experimental autoimmune thyroiditis, in a manner similar to what is observed in Hashimoto's thyroiditis in humans.

Actin Cytoskeleton Is Required for Early Apoptosis Signaling Induced by Anti-Fas Antibody but Not Fas Ligand in Murine B Lymphoma A20 Cells

Murine B lymphoma A20 cells are highly sensitive to Fas-mediated death signals induced by anti-Fas antibody Jo2 or cross-linked Fas ligand (FasL). We have found that the microfilament poison cytochalasin D blocks Fas-mediated apoptosis induced by Jo2 but not FasL in A20 cells. The induction of Fas-mediated apoptosis by Jo2 was antagonized by anti-Fcγ RII/RIII receptor (FcγR) antibody, and defective in FcγR-negative A20 cells. Since the induction of Jo2-mediated apoptosis in FcγR-negative A20 cells was reversed by the addition of wild type A20 cells or the cross-linking agent protein A, Fas-expressing bystander A20 cells seem to be killed by other A20 cells that capture and cross-link monomeric Jo2 via FcγR. Although cytochalasin D affected FcγR-mediated cross-linking of Jo2 molecules, the drug markedly inhibited the intracellular signaling pathway induced by Jo2. The blockade of Jo2-induced apoptosis by cytochalasin D occurred upstream of caspase-8 activation. Thus, these observations suggest that actin cytoskeleton is required for early apoptosis signaling induced by Jo2, but not physiological FasL.

Induction of Salivary Gland Epithelial Cell Injury in Sjogren's Syndrome: In Vitro Assessment of T Cell-derived Cytokines and Fas Protein Expression

Sjogren's syndrome (SS) is an exocrinopathy characterized by T cell infiltrates, salivary gland epithelial cell (SGEC) apoptosis and high Fas and FasL expression. To address the participation of T cell-derived cytokines and of Fas apoptotic pathway in SS glandular lesions, we utilized non-neoplastic SGEC lines established from SS patients and controls. Possibly attesting to their intrinsic activation, cell lines derived from SS patients displayed significantly higher constitutive Fas and FasL than controls. Surface co-expression of Fas and FasL was not associated with spontaneous fratricide apoptosis. SGEC were resistant to anti-Fas-mediated apoptosis (possibly owing to the constitutive expression of anti-apoptotic proteins cFLIP and Bcl-2), but became sensitive after protein or RNA synthesis inhibition. IFN-γ and TNF-α were able to upregulate surface Fas and FasL, whereas IL-1β downregulated surface FasL. IFN-γ (but not several other cytokines) reduced the survival of SGEC in a dose- and time-dependent manner and induced Fas/FasL-mediated apoptosis, directly and via anoikia. Dexamethasone inhibited the upregulation of Fas and FasL by IFN-γ and the induction of SGEC apoptosis and detachment by anti-Fas mAb or IFN-γ. Our findings indicate the injurious role of IFN-γ for the salivary epithelia of SS patients through the induction of Fas-mediated apoptosis and anoikia.

Amplification of engrafted hepatocytes by preparative manipulation of the host liver.

Scarcity of donor livers is a major obstacle to the general application of hepatocytes for the development of bioartificial liver assist devices as well as intracorporeal engraftment of hepatocytes for the treatment of inherited metabolic diseases. The number of hepatocytes that can be transplanted into the liver safely in a single sitting also limits the utility of this procedure. These limitations could be addressed by providing preferential proliferative advantage to the transplanted cells. Studies using transgenic mouse recipients or donors have indicated that massive repopulation of the host liver by engrafted hepatocytes requires that the transplanted cells are subjected to a proliferative stimulus to which the host hepatocytes cannot respond. Prevention of host hepatocyte proliferation has been achieved by treatment with a plant alkaloid, retrorsine. Because retrorsine is carcinogenic, we have evaluated preparative irradiation for this purpose. The proliferative stimulus may consist of the loss of hepatic mass (e.g., partial hepatectomy, reperfusion injury or induction of Fas-mediated apoptosis by gene transfer) or administration of stimulants of hepatocellular mitosis (e.g., growth factors or thyroid hormone). Potential applications of these preparative manipulations of the host liver include the treatment of inherited metabolic disorders by transplantation of allogeneic hepatocytes, hepatocyte-mediated ex vivo gene therapy, rescuing liver cancer patients from radiation-induced liver damage, and expansion of human hepatocytes in animal livers.

Differential regulation of FAS-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor α and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules

Fas-mediated apoptosis is associated with the pathophysiology of rheumatoid arthritis (RA). However, the molecular mechanisms of this process remain to be elucidated in rheumatoid synovium. We investigated the behavior of intracellular signaling molecules that regulate Fas-mediated apoptosis in RA synoviocytes. Anti-Fas monoclonal antibody (mAb) was added to RA synoviocytes after treatment with tumor necrosis factor alpha (TNFalpha) or basic fibroblast growth factor (bFGF) for 5 days. The cytotoxic activity was measured using a lactate dehydrogenase-release assay. The expression of apoptosis-related molecules in RA synoviocytes was examined by immunoblot analysis. The enzymatic activities of caspases 3 and 8 under Fas ligation were examined. Transfer of the FADD (Fas-associated death domain) protein and the FLIP(L) (long form of the FLICE [FADD-like interleukin-1beta-converting enzyme]-inhibitory protein) gene into RA synoviocytes was performed using adenoviral vectors. Following a 5-day treatment with TNFalpha or bFGF, Fas ligation with its agonistic mAb induced apoptosis of almost all TNFalpha-treated RA synoviocytes but only showed a weak apoptotic activity in bFGF-treated synoviocytes. Although there was no correlation between the induction of Fas-mediated apoptosis and the expression of apoptosis-related molecules among these cells, a high enzymatic activity of caspases 3 and 8 was observed only in the TNFalpha-treated RA synoviocytes after Fas ligation. The bFGF-treated RA synoviocytes were relatively resistant to apoptosis induced by FADD gene transfection, as compared with the TNFalpha-treated synoviocytes. In addition, the expression of FLIP(L), an inhibitory molecule of Fas-mediated apoptosis, was reduced in TNFalpha-treated RA synoviocytes, and the expression of FLIP43 was augmented in bFGF-treated RA synoviocytes. Moreover, Fas-mediated apoptosis in TNFalpha-treated RA synoviocytes was partially inhibited by FLIP(L) gene transfection. Our results suggest that Fas-mediated apoptosis of RA synoviocytes is differentially regulated by TNFalpha and bFGF. In addition, the regulatory mechanisms of apoptosis involve the formation of the death-inducing signaling complex, especially at the level of caspase 8 activation, and this process may be partly associated with FLIP expression.

A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity

Defects in Fas-mediated apoptosis are implicated in autoimmune diseases including rheumatoid arthritis (RA). Although induction of Fas-mediated apoptosis could have therapeutic effects on these diseases, it might cause deleterious effects in liver as Fas ligand or an agonistic anti-murine Fas antibody Jo2 causes severe hepatic injury in mice. We report here on the interesting characteristics of the newly obtained anti-Fas mAb, HFE7A, which cross-reacts with the Fas molecules of various species ranging from human to mouse and mitigates autoimmune symptoms without hepatotoxicity in mice. The administration of HFE7A to mice induced apoptosis in the thymocytes, although administration of HFE7A to mice or to marmosets did not induce any sign of hepatitis. The effect of HFE7A on liver is different from that of anti-murine Fas antibody Jo2, which causes acute and lethal hepatic injury to mice. Administration of HFE7A reduced lymphadenopathy and abnormal T cells in MRL-gld/gld mice. HFE7A induced apoptosis in synovial cells prepared from RA patients. Surprisingly, HFE7A protected mice from fulminant hepatitis induced by Jo2. Therefore, HFE7A is a potential therapeutic antibody not only for autoimmune diseases including RA but also for fulminant hepatitis.

Insulin-like Growth Factor I Stimulates Proliferation and Fas-Mediated Apoptosis of Human Osteoblasts

In vitrostudies have shown that insulin-like growth factor I (IGF-I) is a potent growth factor for osteoblasts, although both bone formation and resorption are upregulated by IGF-Iin vivo.To understand the difference in the action of IGF-I observedin vitroandin vivoexperiments, we examined the effect of IGF-I on the proliferation and Fas-mediated apoptosis of human osteoblastsin vitro.Human osteoblastic cell line MG63 and human primary osteoblast-like cells obtained from biopsy specimens were used as human osteoblasts. Cells were cultured with or without various concentrations of IGF-I followed by determination of the proliferative response and Fas-mediated apoptosis. IGF-I dose dependently stimulated the proliferation of cultured human osteoblasts. Both Fas expression and the degree of anti-Fas IgM-induced apoptosis of human osteoblasts was also augmented by IGF-I. Furthermore, the cytotoxicity of Fas ligand (FasL) cDNA transformants against human osteoblasts was increased when IGF-I-stimulated osteoblasts were used as target cells, indicating that stimulation of IGF-I increased functional Fas expression on human osteoblasts as well as their proliferation. The addition of DEVD-CHO, a specific inhibitor of CPP32, to the culture resulted in a significant inhibition of Fas-mediated apoptosis of both unstimulated and IGF-I-stimulated osteoblasts, although it did not affect the proliferative response or Fas expression. Our data suggest that activation of CPP32 is necessary for Fas-mediated apoptosis of human osteoblasts, and treatment of IGF-I increased this signaling pathway. In contrast, regulation of proliferation and Fas expression of the cells were probably not affected by CPP32 activation. Our results suggest that IGF-I acts on cultured human osteoblasts by increasing their proliferation and induction of Fas-mediated apoptosis by neighbouring FasL+cells such as osteoclasts, thus probably functioning as a local coupling factor in the bonein vivo,stimulating both bone formation and resorption.

The Molecular Interaction of Fas and FAP-1: A TRIPEPTIDE BLOCKER OF HUMAN Fas INTERACTION WITH FAP-1 PROMOTES Fas-INDUCED APOPTOSIS

Fas (APO-1/CD95), which is a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that induces apoptosis. A protein tyrosine phosphatase, Fas-associated phosphatase-1 (FAP-1), that was previously identified as a Fas binding protein interacts with the C-terminal 15 amino acids of the regulatory domain of the Fas receptor. To identify the minimal region of the Fas C-terminal necessary for binding to FAP-1, we employed an in vitro inhibition assay of Fas/FAP-1 binding using a series of synthetic peptides as well as a screen of random peptide libraries by the yeast two-hybrid system. The results showed that the C-terminal three amino acids (SLV) of human Fas were necessary and sufficient for its interaction with the third PDZ (GLGF) domain of FAP-1. Furthermore, the direct cytoplasmic microinjection of this tripeptide (Ac-SLV) resulted in the induction of Fas-mediated apoptosis in a colon cancer cell line that expresses both Fas and FAP-1. Since t(S/T)X(V/L/I) motifs in the C termini of several other receptors have been shown to interact with PDZ domain in signal transducing molecules, this may represent a general motif for protein-protein interactions with important biological functions.

P-129 - Induction of Fas-mediated apoptosis in synovial cells from patients with rheumatoid arthritis

P-129 - Induction of Fas-mediated apoptosis in synovial cells from patients with rheumatoid arthritis

Fas Receptor (CD95)-Mediated Apoptosis in Leukemic Cells

Binding of Fas ligand (FasL) or an agonistic anti-Fas receptor (Fas/CD95) antibody induces apoptosis in Fas-bearing target cells. The involvement of Fas/FasL pathway has been investigated in human acute myelogenous leukemia (AML) cells. Fas/CD95 is expressed on a majority of AML cells, although the intensity of expression is variable. The cross-linking with anti-Fas antibody can induce apoptotic cell death in certain cases of AML. When DNA synthesis and cell cycle progression are enhanced by growth-promoting cytokines, such as interleukin-3 and granulocyte-macrophage colony-stimulating factor. Fas-insensitive AML cells acquire cellular susceptibility toward Fas-mediated apoptosis. Cell cycle analysis reveals that Fas-mediated apoptotic signals can be transduced into cells in G1B compartment and G1A ± G1B transition might support the induction of Fas-mediated apoptosis. In addition, Fas-mediated apoptotic cell death of AML cells is also induced by interleukin-2-activated T cells expressing functional FasL on their surfaces. Activated T cells express a large amount of FasL mRNA, compared with freshly isolated T cells. The Fas/FasL pathway seems to be the major mechanism of T cell-mediated apoptosis in AML cells, although alternative mechanisms can also be operative. The induction of apoptosis in Fas/FasL system might be a novel and effective approach for leukemia immunotherapy.

Induction of Fas-mediated apoptosis on circulating lymphocytes by surgical stress.

The authors determined whether the decrease in lymphocytes after surgery is related to apoptosis. Surgery induces a profound but transient depletion of circulating lymphocytes, However, the mechanism underlying this phenomenon is unclear. Peripheral blood mononuclear cells were obtained from 18 patients before and after elective surgery and studied for morphologic and biochemical markers of apoptosis, DNA fragmentation, and Fas expression. The DNA staining of peripheral blood mononuclear cells obtained after surgery, which had been cultured for 24 hours in vitro, showed chromatin condensation and fragmentation of cells into collapsed spheres. Moreover, DNA isolated from these peripheral blood mononuclear cells formed a ladder of oligonucleosomal fragments. However, peripheral blood mononuclear cells obtained before surgery showed neither of these changes. The observation that none of these apoptotic cells ingested latex suggested that they were of lymphocytic origin. Fas-positive lymphocytes increased significantly 2 hours after the start of surgery and returned to preoperative levels by postoperative day 7. Anti-Fas antibody augmented apoptosis, whereas ZB4, a Fas antagonist, inhibited apoptosis in lymphocytes after surgery. These results indicate that circulating lymphocytes in the early perioperative period are susceptible to Fas-mediated apoptosis, which may cause depletion of circulating lymphocytes after surgery.