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Results for “Fas Ligand Protein”

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At least 19 recordsLinked to original sources
PubMed2001

CD8+ T cells in cutaneous T-cell lymphoma: expression of cytotoxic proteins, Fas Ligand, and killing inhibitory receptors and their relationship with clinical behavior.

PURPOSE: We investigated the number, phenotype, and prognostic significance of CD8+ T cells in patients with mycosis fungoides (MF) and CD30- primary cutaneous large T-cell lymphoma (PCLTCL). PATIENTS AND METHODS: Immunohistochemical stainings for CD8, granzyme B (GrB), T cell-restricted intracellular antigen (TIA-1), Fas ligand (FasL), and killer-cell inhibitory receptors (KIRs; CD95, CD158a, and CD158b) were performed on 83 first-diagnostic biopsy samples obtained from patients with plaque-stage MF (n = 42), tumor-stage MF (n = 20), and CD30- PCLTCL (n = 21). RESULTS: Serial sections and double-staining experiments showed that the large majority of CD8+ T cells in MF and CD30- PCLTCL expressed TIA-1 and FasL, whereas only a minority expressed GrB, which suggested that these CD8+ T cells were partly activated cytotoxic T lymphocytes (CTLs). These CD8+ CTLs never or rarely expressed KIRs. This phenotype was a constant feature of CD8+ CTLs and did not alter with disease progression. In contrast, the median percentage of CD8+ CTLs in plaque-stage MF (22%), tumor-stage MF (7%), and CD30- PCLTCL (3%) differed significantly (P < .0001) and was associated with a significant decrease in 5-year survival. Also within the group of tumor-stage MF, a significant relation between CD8+ CTLs and survival was found. Multivariate analysis in the total group of MF demonstrated that both skin stage and percentage of CD8+ CTLs were independent parameters of survival. CONCLUSION: Our results demonstrated that partly activated CD8+ CTLs were present in CTCL and that high proportions of these cells correlated with a better prognosis. This suggested that these CD8+ CTLs could play an important role in the antitumor response in these conditions.

Adult↗
PubMed1999

Transplacental effects of IgG generated against soluble 53 kDa protein on the splenic lymph system of rat progeny exposed to carcinogen: rate of apoptosis, proliferation of lymphocytes and expression of Fas and Fas ligand proteins.

BACKGROUND: We have previously shown that vaccination with IgG generated against the soluble 53 kDa (s53) protein modified the splenic response to carcinogens. Here we studied whether such immunization could affect the splenic lymphatic system of the offspring. METHODS: Offspring of normal female rats or of rats immunized with anti-s53 IgG were exposed to a carcinogen (dimethyl-benz(a)antracene). After 4 months, their spleens were resected and evaluated immunohistochemically for lymphocyte proliferation, apoptosis and apoptosis-related proteins (Fas and Fas ligand), in tumor-free and tumor-bearing animals. RESULTS: Spleens of progeny of unvaccinated rats had a significant decrease in the areas of follicles, germinal centers and the mantle layer after exposure to carcinogens, while maternal vaccination resulted in a significant expansion of the progeny's splenic follicles and germinal centers, the zones of B cell proliferation. The area of periarterial lymph sheaths (PALS) expanded in these offspring, reflecting activation of the T-zone. Maternal vaccination also resulted in a significant rise of Fas ligand-positive lymphocytes in the follicles and PALS of their tumor-free offspring. Tumorigenesis stimulated the Fas activity of B and T cells in the spleens, and this was much enhanced by maternal vaccination. CONCLUSIONS: Maternal vaccination before pregnancy results in altered morphological and functional attributes of the splenic immune system of the offspring. This increased immunoreactivity could reduce the risk of tumors in progeny of vaccinated mothers.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Apoptosis-related proteins (Fas, Fas ligand, bcl-2 and p53) in different types of human breast tumors.

The aim of this study was to evaluate the possible role of apoptosis-related proteins (ARP: Fas and Fas ligand, bcl-2, p53) in the progress of tumorigenesis in breast cancer. Epithelial tumor cells and lymphocytes were analyzed immunohistochemically for the rate of lymphoid infiltration and presence of ARP in 50 human breast tumors of different types. The tumors included: i) fibrocystic disease (n=12); ii) benign fibroadenoma (n=11); iii) carcinoma in situ (n=8); iv) invasive ductal carcinoma with high lymphoid infiltration (n=12); and v) invasive ductal carcinoma with lymphoid depletion (n=7). Both fibrocystic disease and fibroadenomas had low amounts of lymphocytes and very little lymphoid infiltration. In cancer in situ, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a rise in the total number of lymphocytes, reflecting intensification of the immune response. In carcinomas with high lymphoid infiltration, a significant increase in the number of Fas and FasL and p53-positive cells was found. The number of bcl-2-positive tumor cells in these tumors was not changed, whereas the number of bcl-2-positive lymphocytes decreased. In carcinomas with lymphoid depletion, the opposite picture was found as a result of deep subcompensation of the lymph system. ARP are present in a significantly higher number of lymphocytes than of the epithelial tumor cells. This indicates that the cells initiating apoptosis in tumors are themselves damaged by the process. The intense apoptosis in lymphocytes in malignant tumors may be one of the reasons for the progress of breast tumors.

Apoptosis↗
PubMed2000

Apoptosis and apoptosis-related proteins (Fas, Fas ligand, bcl-2, p53) in macrophages of human ovarian epithelial tumors.

Apoptosis and the apoptosis-related proteins (ARP) (Fas, Fas ligand (FasL), bcl-2 and p53) were analyzed in macrophages of different human ovarian epithelial tumors. Few macrophages were found in ovaries of women without oncologic disorders. In ovarian benign cysts, macrophagic density reached 4.9+/-1.2 per 50,000 microm2, most were present in lymphoid-macrophagic infiltrates of the sub-epithelial stroma (3.7+/-0.5% of the area of a slide), and 23.4% were Fas and FasL positive. In borderline tumors, the expanse of lymphoid infiltrates increased to 15.6% of the area of a slide, and the number of macrophages increased 2.4-fold compared to benign cysts. Of the macrophages, 83-88% expressed Fas and FasL, few had bcl-2 and CD25 receptors, and isolated ones were apoptotic. In carcinomas with high lymphoid-macrophagic infiltration, the infiltrate occupied 17.5% of the slide and macrophages amounted to 12.1+/-1.5/50,000 microm2. Many macrophages were in regions of grouping apoptosis of tumor epithelial cells and significantly fewer expressed Fas, FasL and bcl-2. Macrophages destroyed by apoptosis accounted for 4.6%. In carcinomas with low lymphoid-macrophageal infiltration, the area of the last was 5.1% of the slide. There were 8.6+/-0.8 macrophages/50,000 microm2, mainly at the margins of zones of necrosis and of tumor cells' grouping apoptosis. Extensive macrophagic infiltration into tumor parenchyma is one way by which the host immune system destroys tumors. Fas and FasL appear in macrophages of benign cysts, but in borderline tumors and in carcinomas with low infiltration their concentration increases sharply, to 79.8% and 96.6%, respectively. In 4.5% of these cells, apoptosis of macrophages was seen. The findings suggest that macrophages participate in the transfer of ARP to tumor epithelial cells, thereby inducing their apoptosis, but undergoing the simultaneous apoptosis.

Aged↗
PubMed2003

Display of Fas ligand protein on cardiac vasculature as a novel means of regulating allograft rejection.

BACKGROUND: Fas ligand (FasL) is a potent death-inducing molecule with important functions in immune homeostasis and tolerance to self-antigens. The complex biological activities of FasL and its inefficient expression using conventional gene transfer approaches limit its use for immunomodulation to prevent allograft rejection. We have recently generated a chimeric FasL with core streptavidin (SA-FasL) with potent apoptotic activity and designed a novel approach to display it on the surface of several cell types via biotinylation. We herein tested whether SA-FasL can also be displayed on vascular endothelial cells in the heart and examined its effect on graft survival after transplantation into syngeneic and allogeneic hosts. METHODS AND RESULTS: SA-FasL was efficiently displayed on the vasculature of BALB/c hearts with a half-life of 9 days in vivo. Transplantation of hearts displaying SA-FasL into syngeneic hosts resulted in indefinite graft survival without detectable toxicity to the grafts and hosts. In contrast, transplantation of allogeneic C57BL/10 hearts displaying SA-FasL into BALB/c recipients resulted in graft rejection, but in a delayed fashion as compared with control hearts (mean survival time=17.4+/-5 versus 9.6+/-1 days). Allograft survival was further extended to 21+/-2.6 and 24+/-3 days (P<0.05) by intravenous treatment of graft recipients with 1 dose of SA-FasL-decorated donor splenocytes on days 2 and 6 after transplantation, respectively. CONCLUSIONS: This study shows for the first time that exogenous proteins can be displayed on the endothelium of solid organs for therapeutic purposes. This approach provides a convenient and rapid means of displaying exogenous proteins on the surface of cells, tissues, and solid organs, with broad research and therapeutic implications.

Animals↗
PubMed2001

Lymphoid elements and apoptosis-related proteins (Fas, Fas ligand, p53 and bcl-2) in lichen sclerosus and carcinoma of the vulva.

We studied some of the morphological and immunohistochemical parameters of lichen sclerosus (LS) and carcinomas of the vulva in order to verify some characteristics in LS related to neoplasm transformation. Parameters such as proliferating index, rate of proliferation of lymphoid elements into a tumor and types of such elements were studied. In parallel, the number of cells positive to apoptosis-related proteins such as Fas, Fas ligand, p53 and bcl-2 were evaluated. Biopsy material from patients with different vulvar disorders--22 samples with LS and 23 samples with vulvar squamous cell carcinoma (VSCC)--was studied by the methods of morphometry and immunohistochemistry. In LS, the number of T cells is a few times higher than those of B cells. Among the T cells, the number of killers is significantly higher than the number of helpers. Carcinomas, especially those with lymphoid depletion, are characterized by a further significant increase in some parameters such as the rate of lymphoid proliferation and the number of T helpers and killers. The progression in to tumorigenesis was accompanied with a significant increase in the number of Fas+ and FasL+ lymphocytes. In tumor epithelial cells the proliferative index increased in carcinomas with lymphoid depletion. The number of p53+ epithelial cells increased whereas the number of bcl-2+ cells showed a distinct tendency to decrease with progression in to tumorigenesis. Development of a tumor is manifested in deep changes in relationships between different lymphoid components. Only two lymphoid markers are significantly different in VSCC compared to LS: the number of T killers and macrophages. The other parameters studied (rate of proliferative activity, the total number of T cells and T helpers, B cells, IL-2-connective cells) already showed high expression in LS as the first signs of transformation of this inflammation into neoplasia.

Apoptosis↗
PubMed1999

Soluble Fas and soluble Fas ligand proteins in human milk: possible significance in the development of immunological tolerance.

Human milk contains a complex uncharacterized, immune system able to exert actions both locally and systemically. This study reports the results of an ELISA-based quantitation of soluble Fas and soluble Fas ligand in human milk which may modulate the Fas/FasL system that is critical for the expression of immune tolerance and apoptosis. Production of Fas/FasL mRNAs by milk cells was also examined using RT-PCR. Fas is ubiquitously expressed in various cells and when bound by its ligand FasL, present predominantly on activated T- and NK cells, Fas-expressing cells are killed. A large amount of soluble Fas (1746-4320 pg/ml) is detected in colostrum, transitional milk and the mature milk of mothers delivering prematurely or at full-term, whereas FasL is present only in the range 123-310 pg/ml. Milk cells are positive for Fas mRNA, but negative for FasL mRNA. An excess of soluble Fas in human milk may bind to FasL preventing apoptosis and preserving epithelial barriers, and may represent an additional new mechanism whereby human milk favours immune tolerance and normal gastrointestinal development.

Colostrum↗
PubMed2003

Fas and Fas ligand protein and mRNA in normal and atretic mouse ovarian follicles.

Apoptosis is the underlying mechanism of follicular atresia in the mammalian ovary. However, the apoptotic pathways governing this ovarian process are not completely elucidated. In the present study, expression of Fas and Fas ligand, the proximal members of the death receptor pathway, was evaluated in mouse ovarian follicles using immunofluorescence and in situ hybridization. Normal or atretic follicles were obtained from immature female Swiss mice after administration of 10 iu equine chorionic gonadotrophin for 48 or 72 h, respectively. Expression of both Fas and Fas ligand mRNA and protein was observed in granulosa cells of normal and atretic follicles. Although the oocytes of normal follicles failed to show any staining, those of atretic follicles stained intensely for Fas, indicating that the presence of Fas in the oocyte determines the fate of the follicle.

Animals↗
PubMed2003

Expression of Fas and Fas ligand protein and mRNA in mouse oocytes and embryos.

During mammalian embryonic development, abnormal eggs and embryos are eliminated by apoptosis; however, the precise apoptotic pathways remain as yet unidentified. In the present study, expression of Fas and Fas ligand - the proximal members of the death receptor pathway, was evaluated in mouse preimplantation embryos by immunofluorescence and in situ hybridization techniques. Ovulated oocytes were collected from oviducts of cyclic mice on the day of oestrus (day 0), and one-cell, two-cell embryos and eight-cell morulae were collected from oviducts of mated animals on days 1, 2 and 3 of pregnancy, respectively. Blastocysts were flushed from the uterine horns on day 4. Expression of Fas and Fas L mRNAs and proteins was absent from embryos at days 0, 1 and 2. A marked increase in Fas and Fas L mRNA and protein expression was detected in all morphologically normal embryos on day 3 and day 4. In addition, embryos on days 3 and 4 were positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining; however, absence of caspase 8 and 3 and intense localization of proliferating cell nuclear antigen confirmed the proliferative status of these embryos. Furthermore, TUNEL staining was absent in postimplantation embryonic sections obtained on day 6. The results of the present studies thus indicate an equilibrium between proliferation and apoptosis in the preimplantation embryo.

Animals↗
PubMed2002

The expression of Fas ligand protein in ultraviolet-exposed rabbit corneas.

PURPOSE: Keratocytes undergo apoptosis during various pathologic conditions and after exposure to ultraviolet radiation (UVR). It was reported that the Fas/Fas ligand system is involved in modulating keratocyte apoptosis. The expression of Fas ligand (FasL) protein was studied in rabbit corneas after photokeratitis induced by different UV wavelengths. METHODS: Six New Zealand albino rabbit corneas were exposed to 280- and 310-nm UVR in 10-nm full wavebands at doses producing biomicroscopically significant keratitis (0.12 J/cm2 for 280 nm and 0.47 J/cm2 for 310 nm). Animals were killed 24 hours after exposure. Immunohistochemistry was performed to localize FasL protein in paraffin sections of rabbit corneas. Primary antibody was polyclonal goat anti-FasL IgG. RESULTS: FasL protein was uniformly detected in epithelial and endothelial layers of all UVR-exposed and control, nonexposed corneas. The positive staining of keratocytes was confined to the anterior stroma of corneas that were exposed to 280-nm UVR. Corneas exposed to 310-nm UVR showed positively stained keratocytes throughout the entire thickness of the stroma. CONCLUSIONS: These data strongly suggest that the Fas/FasL system may play an important role in apoptosis of corneal cells after UVR. The FasL expression in the corneal stroma was more extensive after exposures at 310-nm UVR than at 280-nm UVR.

Animals↗
PubMed2000

Apoptosis and apoptosis-related proteins (Fas, Fas ligand, Blc-2, p53) in lymphoid elements of human ovarian tumors.

Different types of lymphocytes have different roles in tumor suppression. Thus, their expression of apoptosis-related proteins (ARP - Fas and Fas ligand, bcl-2, p53) in lymphocytes and their apoptosis were analyzed immunohistochemically in ovarian tumors of different grades. Ovaries without oncologic disorders had few lymphocytes, mainly T cells, and no ARP. Benign cysts presented features of weak immune reaction: small lymphoid infiltration and few lymphocytes. The ARP were present in 13.7% to 23.5% of the lymphocytes, and apoptosis was rare. In borderline tumors, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a tenfold rise in total lymphocytes, reflecting intensification of the immune response. Most lymphocytes were T cells (92%) predominated by CD8+ cells that were in direct contact with tumor epithelial cells. ARP species were found in 47% to 65% of the lymphocytes, and apoptosis in 2.2%. In carcinomas with ligh lymphoid infiltration, lymphocytes were 2.5 times more abundant, and the apoptotic index as well as the number of CD20+ and CD25+ lymphocytes rose sharply, whereas bcl-2 positive lymphocytes decreased to 8% of their number in borderline tumors. In carcinomas with low lymphoid infiltration, the total lymphocyte count decreased eightfold compared to carcinomas with high lymphoid infiltration, reflecting the deep subcompensation of the lymphoid system. Few p53-positive lymphocytes were found in the carcinomas. In conclusion, we found a positive correlation between apoptosis and the numbers of CD4+ or CD8+ lymphocytes in epithelial ovarian tumors. This correlation could reflect the antitumor activity of T cells. However, the high expression of ARP studied by immune cells at the vicinity of the tumor ARP reveals the lymphoid vulnerability to apoptosis, resulting in devastation of the lymphoid tissue, and consequently in tumor progression.

Adenocarcinoma↗
PubMed1997

Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense.

In vertebrate tissues, cell integrity is maintained by at least three mechanisms. During an immune response, injured cells are eliminated by cytotoxic lymphoid cells that produce perforin, granzyme B, and Fas ligand (FasL). Second, epithelial cells can produce FasL as an immunosuppressive protein, probably to protect the tissue against immune-mediated damage. Third, locally secreted antimicrobial peptides can be operative in the protection of animal and human epithelia. In this work, as another contribution to local mechanisms of host defense, the ability of human epidermal keratinocytes to produce cytotoxic proteins was investigated. To address this question, freshly isolated human epidermal cells and keratinocytes grown in vitro were studied. Freshly isolated epidermal cells did not express the cytolytic proteins. In contrast, keratinocyte growth to confluence was associated with granzyme B, perforin, and FasL mRNA and protein synthesis. These proteins were secreted in the culture medium. Further analysis showed that they were identical with the ones used by cytotoxic lymphocytes. Their function was then investigated with a view to a potential role in epidermal cell integrity. The data showed that activated human keratinocytes were able to protect against invading pathogens through granzyme B expression. This was demonstrated by the ability of granzyme B to greatly decrease the bacterial growth of Staphylococcus epidermidis. In addition, keratinocytes expressing FasL were found to prevent immune epidermal cell damage. Apoptosis of Fas-sensitive T cells occurred during coculture with confluent epidermal keratinocytes and was largely reduced by the addition of a FasL inhibitor. The data favor keratinocyte involvement in the regulation of dermal inflammatory responses.

Apoptosis↗
PubMed2002

Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells.

BACKGROUND/AIMS: Increased susceptibility of the kidney to acute renal failure (ARF) in the setting of sepsis even in the absence of systemic hypotension is well known. In the hypothesis that the proinflammatory cytokines and lipopolysaccharide (LPS) in gram-negative sepsis can directly cause renal tubular cell apoptosis via Fas- and caspase-mediated pathways, we examined apoptosis and Fas, Fas ligand, FADD expression, as well as PARP cleavage in cultured human proximal tubular cells under the cytokine and LPS-stimulated conditions. METHODS: HK-2 cell, immortalized human proximal tubular cell lines, were treated with 5 and 30 ng/ml of tumor necrosis factor-alpha (TNF-alpha), 5 and 20 ng/ml of interleukin-1beta (IL-1beta) and 30 ng/ml LPS for 24 h. Fas expression was examined by RT-PCR and Fas ligand, Fas-associated protein with death domain (FADD) and poly ADP ribose polymerase (PARP) cleavage were examined by Western blot analysis. Apoptosis was assessed by flow cytometer using Annexin V-FITC and propidium iodide (PI) staining and also by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods. RESULTS: Fas mRNA expression (ratio of Fas/L-19) increased in the TNF-alpha 5, 30 ng/ml and LPS treated group (p < 0.01, p < 0.01, p = 0.02), but there was no difference between the low- and high-dose TNF-alpha groups. Fas ligand protein expression did not increase in the low-dose TNF-alpha treated group, but it increased significantly in the high-dose TNF-alpha treated group (p < 0.01), IL-1beta- and LPS-treated groups (p < 0.01, p = 0.01, p < 0.01, p = 0.02). The intracellular adaptor protein, FADD expression also increased significantly in the high-dose TNF-alpha- and IL-beta-treated groups (p = 0.04, p = 0.04), but in the low-dose TNF-alpha and IL-beta treated group, it did not show statistically significant differences. In the LPS group, FADD expression also showed an increased tendency, but it was not statistically significant (p = 0.09). Western blot for PARP, a DNA repair enzyme mainly cleaved by caspase 3, showed increased 89- and 24-kD PARP cleavage products in TNF-alpha, IL-1beta and LPS treated cells. The degree of apoptosis examined by DNA fragmentation and translocation of membrane phosphatidyl serine significantly increased in cytokines and LPS treated groups. CONCLUSION: These results suggest that Fas- and caspase-mediated apoptosis of tubular cells by inflammatory cytokines and LPS can be one of the possible mechanisms of renal dysfunction in endotoxemia.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Soluble Fas and Fas-ligand in bladder cancer in vitro and in vivo.

Circulating soluble Fas (sFas) and expression of Fas-ligand on cancer cells are mechanisms of immune escape. The aim of the present study was to investigate expression and production of Fas and Fas-ligand on bladder cancer cell lines of different grade as a basic mechanism of their secretion in vivo. sFas and sFas-ligand serum levels of patients with different stage of bladder cancer were examined to determine the possible clinical use of these molecules as tumor markers. Bladder cancer cell lines RT4 (G1), RT112 (G1), T24 (G3) and SUP (G4) were analyzed by flowcytometry for Fas and Fas-ligand expression. To determine if the Fas-ligand gene is transcribed in these bladder cancer cell lines, RT-PCR was performed on mRNA extracted from these cell lines. Production of sFas and sFas-ligand was examined in cell culture supernatants of the cancer cells as well as in the serum of 62 patients with bladder cancer by a specific ELISA test. We demonstrate that Fas is expressed in similar levels on all human bladder carcinoma cell lines. In T24 (G3) and SUP (G4) cell lines we were able to detect the Fas-ligand protein, whereas no Fas-ligand protein could be found in RT4 and RT112 (G1) cells. Fas-ligand mRNA was expressed in all bladder cancer cell lines. Furthermore, all bladder cancer cell lines produce sFas but no sFas-ligand in spite of mRNA expression. The range of sFas levels in the serum of all patients with bladder cancer was large and did not show a correlation to the histopathological stage of bladder cancer. Although there is in vitro evidence that sFas and Fas-ligand play a role in bladder cancer, no correlation between the sFas and s Fas-ligand serum levels and the histopathological stage of bladder cancer could be found. Therefore, serum sFas and sFas-ligand have to date limited clinical relevance.

Journal Article↗
PubMed1997

Characterization of Fas (Apo-1, CD95)-Fas ligand interaction.

The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.

Amino Acid Sequence↗
PubMed2003

Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells.

Antigen-mediated activation of mast cells results in Ca2+-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca2+ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response.

Animals↗
PubMed1997

Activity of TNF-related apoptosis-inducing ligand (TRAIL) in haematological malignancies.

T-cell cytotoxicity is primarily mediated by two cell surface proteins, Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and intracellular perforin and granzyme granules. FasL-deficient and perforin-deficient T lymphocytes maintain cytotoxicity but fail to induce graft-versus-host disease (GVHD) when transplanted into mice. suggesting that GVHD and graft-versus-tumour (GVT) effects can be dissociated, and that TRAIL is not involved in the pathogenesis of GVHD. Because TRAIL could mediate a favourable GVT effect it became important to study the spectrum of its activity and to investigate factors that can dissociate its expression from FasL. TRAIL induced apoptosis in 11/41 (27%) tumour specimens of haematological origin compared to 16/41 (39%) induced by FasL. Although eight specimens were sensitive to both FasL and TRAIL, no synergism was observed between these two ligands. TRAIL induced apoptosis in a dose and time dependent manner with an ED50 of 0.5 microg/ml and EDmax of 1 microg/ml. TRAIL activity was not reduced by the over-expression of the multidrug resistant (MDR) protein, and was not enhanced by 9-cis retinoic acid (RA), which can down-regulate bcl-2 protein. Both ligands were simultaneously up-regulated in normal peripheral blood lymphocytes in response to IL-2, IL-15 and anti-CD3 antibody, whereas IL-10 had no effect. Together, our data show that (1) TRAIL can mediate cell death in a variety of human haematological malignancies, (2) resistance to TRAIL is not mediated by MDR protein, (3) the lack of synergy between TRAIL and FasL suggests that either one is sufficient to mediate T-cell cytotoxicity, and (4) within the panel of cytokines tested, the expression of TRAIL and FasL could not be dissociated.

Apoptosis↗
PubMed1998

Evidence for tight coupling of gonadotropin-releasing hormone receptor to stimulated Fas ligand expression in reproductive tract tumors: possible mechanism for hormonal control of apoptotic cell death.

Fas ligand induces cell death by means of apoptosis in a variety of cell types when cross-linked with its natural receptor, Fas. GnRH receptor-bearing tumors undergo apoptosis in vivo and in vitro with GnRH agonists. To provide a potential association of the Fas system with the antiproliferative signaling process of GnRH receptor, we have evaluated the regulation of Fas ligand expression in GnRH receptor-positive tumors and cloned cell lines known to have substantial GnRH receptors. Surgically removed uterine endometrial carcinomas and ovarian carcinomas had been screened for GnRH receptor expression before analysis. Fas ligand protein was characterized by immunoblotting of membrane proteins with the specific antibody. Fas ligand messenger RNA was determined by RT-PCR using oligonucleotide primers synthesized according to the published Fas ligand sequence. Incubation with a GnRH analog (1 mumol/L) induced the expression of Fas ligand messenger RNA and immuno-reactive Fas ligand with a lag time of 48 h in cloned cell lines (endometrial carcinoma HHUA cells, and ovarian carcinoma SK-OV-3 and Caov-3 cells). There was no detectable Fas ligand expression within 24 h. The stimulatory effect of GnRH on Fas ligand protein expression revealed a dose dependency; a half-maximal effect occurred with 10 nmol/L GnRH analog (P < 0.01). The stimulated Fas ligand expression could be neutralized by displacement of GnRH from its receptor by GnRH antagonist antide. Cells isolated from GnRH receptor-bearing ovarian carcinomas and uterine endometrial carcinomas gave identical results to those obtained in cloned cell lines. These data demonstrate the functional coupling of stimulated Fas ligand expression to GnRH receptor activation. Increased Fas ligand level within the GnRH receptor-bearing tumors might promote apoptotic cell death through attack on intratumoral Fas-positive cells that could, at least in part, account for the antiproliferative action of the hormone.

Apoptosis↗