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1.
Motomu Shimizu Yasutaka Takeda Hideo Yagita Takayuki Yoshimoto Akio Matsuzawa 《Cancer immunology, immunotherapy : CII》1998,47(3):143-148
Lymph node (LN) cells of Fas-mutant mice lpr/lpr (lpr) and lpr
cg
/lpr
cg
(lpr
cg
) express an increased level of Fas ligand (FasL) (CD95L). We examined the antitumor potential of cell-bound FasL on these
LN cells against Fas+ tumor cells. Fas+ F6b and Fas− N1d cells were produced from murine hepatoma MH134 (Fas−) by gene transfection. lpr and lpr
cg
LN cells inhibited growth of F6b but not N1d cells in vitro. Neither gld/gld lpr/lpr (gld/lpr) LN cells, which lack both FasL and Fas, nor wild-type LN cells showed growth-inhibitory activities against F6b and N1d cells.
The effector cells and molecule were CD4−CD8− T cells and FasL, respectively. The tumor neutralization test and adoptive transfer demonstrated that lpr and lpr
cg
, but not gld/lpr, LN cells retarded the growth of F6b cells. Although anti-Fas antibody and FasL cause severe liver failure, wild-type mice
injected with lpr LN cells appeared clinically normal. Adoptive transfer of lpr LN cells to F6b-bearing mice exerted the same antitumor activity in wild-type and gld/lpr recipient mice, indicating the applicability of cell-bound FasL for Fas-mediated target therapy of cancer. These results
suggest that antitumor activity was dependent on the Fas-FasL system and that lymphoid cells overexpressing FasL can be powerful
antitumor effector cells against Fas+ tumor cells.
Received: 16 March 1998 / Accepted: 28 July 1998 相似文献
2.
G. Gradl P. Grandison E. Lindridge Y. Wang J. Watson F. Rudert 《Apoptosis : an international journal on programmed cell death》1996,1(2):131-140
Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown
previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine
L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino
acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291)
and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also
mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated
proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein
species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent
a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex,
and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction.
This work was in part supported by a grant from the Health Research Council of New Zealand (to JW). 相似文献
3.
The anti-cancer drug etoposide can induce caspase-8 processing and apoptosis in the absence of CD95 receptor-ligand interaction 总被引:1,自引:0,他引:1
Boesen-de Cock JG de Vries E Williams GT Borst J 《Apoptosis : an international journal on programmed cell death》1998,3(1):17-25
Caspase-8 (FLICE) can associate with and be activated by CD95 (APO-1/Fas), an apoptosis-inducing member of the Tumour Necrosis Factor receptor family. We find that, in Jurkat T cells, the DNA damaging anti-cancer drug etoposide induces apoptosis and, surprisingly, processing of caspase-8. Therefore, we have investigated whether etoposide involves CD95 receptor activation. We find that etoposide does not induce CD95 ligand expression at the mRNA level. In addition, blocking of CD95 receptor function with a specific antibody does not inhibit etoposide-induced apoptosis. Apparently, in Jurkat cells, etoposide can induce caspase-8 processing and apoptosis in a CD95-independent fashion. Likewise, we find that thymocytes from the CD95-deficient lpr/lpr mouse strain readily undergo apoptosis in response to etoposide. Moreover, since inhibition of the secretory pathway with brefeldin A does not inhibit etoposide-induced apoptosis, we exclude the requirement for a newly synthesizedreceptor ligand to induce the apoptotic pathway. We conclude that, at least in certain cell types, etoposide does not require CD95 receptor function to induce caspase-8 processing and apoptosis. 相似文献
4.
5.
Expression and characterization of biologically active human Fas ligand produced in CHO cells 总被引:1,自引:0,他引:1
Zappitelli S D'Alatri L Ciucci A Raucci G Faiella A Gabrielli M Parlani M Bressan A Maggi CA Goso C Rotondaro L 《Molecular biotechnology》2003,23(3):189-202
We describe an expression system for high-yield production of recombinant soluble human FasL (rsh-FasL) in CHO cells. After
one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 μg/L × 106 cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into
growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a
form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while,
although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of
native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive
cells and in in vitro binding assays.
S. Zappitelli and L. D’Alatri contributed equally to this work. 相似文献
6.
Death ligands such as CD95 ligand (CD95L) have limited activity against glioma cells under normoxic conditions. Hypoxia is a critical aspect of the microenvironment of gliomas in vivo. We investigated the effect of co-exposure to acute hypoxia and CD95 ligand in three human malignant glioma cell lines with different susceptibility to CD95L under normoxic conditions. Hypoxia sensitized all three cell lines towards CD95L-induced cell death. Co-exposure resulted in apoptotic changes in the early phase, with gradual conversion to secondary necrosis with increasing length of hypoxia. The mitochondrial injury induced by hypoxia was enhanced by co-treatment, and caspase cleavage became prominent. Inhibition of the epidermal growth factor receptor (EGFR), although sensitizing glioma cells to CD95L under normoxia, protects glioma cells from hypoxia by reducing energy consumption. However, the opposing effects of EGFR signalling on death induced by CD95L or hypoxia were neutralized by co-exposure to hypoxia and CD95L. Furthermore, inhibition of protein synthesis by cycloheximide also reduced glucose consumption and conferred protection from hypoxia, but did not modulate CD95L-induced cell death under hypoxic conditions. These results suggest that death ligands may be useful to target hypoxic tumour cells resistant to conventional therapies or to complement strategies aiming at the induction of tumour hypoxia. 相似文献
7.
Schmitz I Krueger A Baumann S Kirchhoff S Krammer PH 《Biochemical and biophysical research communications》2002,297(3):459-462
The CD95 (APO-1/Fas) death receptor plays an important role in many physiological and pathophysiological systems. Thus, the CD95 system contributes to activation-induced cell death. Therefore, reliable antibodies recognizing human CD95 are of great interest. Detection of CD95 expression often relies on antibodies, e.g., suitable for Western blotting. To detect CD95, we compared the specificity of nine different anti-human CD95 antibodies recognizing different epitopes by using postnuclear supernatants of four different cell lines. Only two of the antibodies tested, both directed against intracellular epitopes of human CD95, detected endogenous human CD95 by Western blotting. Therefore, we conclude that results obtained with other anti-CD95 antibodies should be treated with caution. 相似文献
8.
Steinbach JP Eisenmann C Klumpp A Weller M 《Biochemical and biophysical research communications》2004,321(3):524-530
Inhibition of epidermal growth factor receptor (EGFR) signaling sensitizes human malignant glioma cells to death ligand-induced apoptosis. However, tumor cells may compensate the loss of EGFR signaling by activation of the type 1 insulin-like growth factor receptor (IGF-1R). We here report that antagonism of the IGF-1R with the small-molecule inhibitor AG1024 in combination with inhibitors of the EGFR synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis. This cell death is p53-independent, but requires caspase 8 activity. The levels of the receptor, CD95, are not altered by the inhibitors alone or in combination. Analysis of the downstream signaling pathways reveals synergistic inhibition of ribosomal protein S6 phosphorylation by inhibitor co-treatment, suggesting an involvement of the mammalian target of rapamycin pathway. These findings suggest that adding inhibitors of IGF-1R may be a strategy to overcome escape from the anti-apoptotic effects of EGFR inhibition in malignant gliomas. 相似文献
9.
Fas (CD95/Apo-1) exists both in membrane-bound and in biologically active soluble (s) forms. Ligation of membrane-expressed
Fas can induce apoptosis, and Fas-mediated signaling seems to be involved in T-cell-induced apoptosis of human acute myelogenous
leukemia (AML) blasts. The local release of sFas by AML blasts may then function as a protective mechanism by competing with
membrane-bound Fas for binding sites on the common Fas ligand (FasL). sFas was released by AML blasts during in vitro culture,
and this release was modulated by several cytokines that can be secreted by activated T cells. Increased levels of sFas could
be detected during in vitro activation of T cells in the presence of native AML accessory cells, and this was observed both
for (i) mitogenic activation of CD4+ and CD8+ T cell clones derived from acute leukemia patients with therapy-induced leukopenia and (ii) allostimulated activation of
T cells derived from normal donors. However, local in vivo levels of sFas will also be influenced by variations in systemic
levels. High serum levels of sFas were detected in acute leukemia patients during chemotherapy-induced cytopenia, but these
levels decreased during complicating bacterial infections. In contrast, serum levels of sFasL were normal in leukopenic patients.
The present results support the hypothesis that local release of sFas can function as a protective mechanism against AML-reactive
T cells, but the effects of this local release are, in addition, modulated by variations in systemic levels of sFas (but not
sFasL).
Received: 9 March 2000 / Accepted: 25 May 2000 相似文献
10.
The potential role of cysteine cathepsins, especially cathepsin B, in Fas/CD95-induced apoptosis was investigated using wild-type and cathepsin B-deficient primary skin fibroblasts. Apoptosis was induced with an anti-Fas/CD95 antibody in the presence of cycloheximide and no difference was observed between the two genotypes. First cells with damaged mitochondria were observed approximately 3h post apoptosis induction and their number was significantly increased after 11h. In contrast, cells with damaged lysosomes were only seen after 15h with no difference between the two genotypes. Moreover, Bid cleavage was found to be diminished in cathepsin B-deficient cells. These results suggest that cysteine cathepsins have no active role in Fas/CD95 apoptosis. 相似文献
11.
CD95/Apo-1/Fas: independent cell death induced by doxorubicin in normal cultured cardiomyocytes 总被引:1,自引:0,他引:1
Doxorubicin is a commonly used cytotoxic drug for effective treatment of both solid tumors and leukemias, which may cause severe cardiac adverse effects leading to heart failure. In certain tumor cells, doxorubicin-induced cell death is mediated by death receptors such as CD95/Apo-1/Fas. Here we studied the role of death receptors for doxorubicin-induced cell death in primary neonatal rat cardiomyocytes and the embryonic cardiomyocytic cell line H9c2.1. Doxorubicin-induced cell death of cardiomyocytes was associated with cleavage of caspases 3 and 8, a drop in mitochondrial transmembrane potential, and release of cytochrome c. Doxorubicin-treated cardiomyocytes secreted death-inducing ligands into the culture supernatant, but remained resistant toward cell death induction by death receptor triggering. In contrast to the chelator dexrazoxane, blockade of death receptor signaling by stable overexpression of transdominant negative adapter molecule FADD did not inhibit doxorubicin-induced cell death. Our data suggest that cultured cardiomyocytes secrete death-inducing ligands, but undergo death receptor–independent cell death upon exposure to doxorubicin.This work was supported by Wilhelm Sander Stiftung and Bettina-Bräu-Stiftung. 相似文献
12.
13.
Fujita H Koji T Kitagawa N Tsutsumi K Abe K Kaminogo M Shibata S 《Cellular and molecular neurobiology》2002,22(4):393-406
1. For a better understanding of the biological features of astrocytic tumors, we investigated apoptosis and its pathway, especially in the interaction between Fas and Fas ligand (FasL).2. We examined the presence of apoptosis in human astrocytic brain tumors by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) and then apoptotic index (AI) was calculated. We also examined the distribution of Fas and FasL-positive tumor cells immunohistochemically. Labeling index (LI) for Fas and FasL was calculated as Fas-LI and FasL-LI, respectively, and compared to AI.3. Tumor cells expressing both Fas and FasL were TUNEL positive. Such cells were distributed sparsely in low-grade astrocytomas, but focally in glioblastomas. There was a close correlation among AI, Fas-LI, and FasL-LI, and astrocytic tumors with higher AI were associated with a longer survival time than that with lower AI.4. It was concluded that the Fas system may be involved in the apoptosis of astrocytic tumors, and AI can be a useful parameter for assessing prognosis of astrocytic tumors. 相似文献
14.
Modification of tumor cells with Fas (CD95) antigen gene and Fas ligand (CD95L) gene transfection by electroporation for immunotherapy of cancer 总被引:1,自引:0,他引:1
Electroporation is a method for introducing DNA into cells by using a high-voltage electric field. This method is very simple
and easily manipulated. We describe here a method for the modification of tumor cells with the Fas/Apo-1 (CD95) antigen-gene
and Fas ligand (FasL)-gene transfection through the use of electroporation, and suggest that the Fas-FasL system is a good
target for the induction of apoptosis-mediated antitumor activity. The Fas receptor/ligand system induces apoptosis and plays
an important role in regulation of the immune system. In the method described, hepatoma MH134 (Fas− and FasL−) is transfected with murine Fas and FasL cDNA. A single administration of monoclonal anti-Fas antibody efficiently suppresses
the growth of F6b (MH134+Neo+Fas) tumors but not that of N1d (MH134+Neo) tumors in gld/gld lpr/lpr mice. MH134+Neo+FasL tumor cells were rejected after the induction of inflammation with infiltration of neutrophils in mice.
These results suggest that electroporation and Fas-mediated apoptosis are a good method for inducing of antitumor activity. 相似文献
15.
Nat R Voiculescu B Stanciu C Vidulescu C Cergan R Badiu C Popescu LM 《Journal of cellular and molecular medicine》2001,5(2):179-187
We analysed the spatial and temporal distribution of apoptosis in human cerebellum development, during embryonic and fetal periods. Cerebella excised from two human embryos (8 weeks old) and eight fetuses (12-22 weeks old), were paraffin embedded and serially sectioned. Apoptotic cells were identified by propidium iodide staining, and TUNEL. In addition, immunohistochemistry for suicide receptor Fas(APO-1/CD95) was performed. We determined the distribution and percentage of apoptotic cells as well as Fas(APO-1/CD95)-positive cells in different regions and stages of development. Apoptotic cells were seen in both proliferative zones and postmitotic regions along the migratory pathways as well as in the developing cerebellar cortex in all examined stages. The Fas(APO-1/CD95) immunoreactivity was present in all examined stages in a small population of apoptotic cells: either neuroblasts or differentiated cells in postmitotic zones. These findings suggest that apoptosis drives the selection of the cells which are committed to differentiate during the early stages of cerebellar development. The differences between apoptotic cells distribution and Fas receptor expression suggest that cell selection is driven by different apoptotic pathways. 相似文献
16.
Characterization of the lipolytic pathways that mediate free fatty acid release during Fas/CD95-induced apoptosis 总被引:1,自引:0,他引:1
Iturralde M Pardo J Lacasa E Barrio G Alava MA Piñeiro A Naval J Anel A 《Apoptosis : an international journal on programmed cell death》2005,10(6):1369-1381
We have undertaken a study to characterize the lipolytic pathway responsible for the generation of free fatty acids (FFA)
during Fas/CD95-induced apoptosis in Jurkat cells. It was initially shown that the cellular lipid fraction that suffered the
major quantitative decrease during Fas-induced apoptosis was that of phosphatidylcholine (PC). In addition, the secretion
of palmitic acid-derived FFA was largely prevented by D609, an inhibitor of PC-specific phospholipase C (PC-PLC) and also
by the diacylglycerol lipase (DAGL) inhibitor RHC-80267, suggesting that the secretion of these FFA during Fas-induced apoptosis
is mediated by the generation of DAG by a PC-PLC activity and, sequentially, by a 1-DAGL activity which generates the FFA
from its sn-1 position. The endocannabinoid 2-arachidonoyl glycerol (2-AG) should be generated as a sub-product of this pathway, but it
did not accumulate inside the cells nor was secreted into the supernatant. Interestingly, the complete inhibition of free
AA secretion during Fas-induced apoptosis was only achieved by using the AA trifluoromethylketone, which not only inhibits
all types of phospholipase-A2 (PLA2) activities, but also the described lytic activities on 2-AG. Using a combination of RHC-80267 and the iPLA2-specific inhibitor bromoenol lactone, it was shown that the DAGL pathway also cooperates with iPLA2 in the generation of free arachidonate. 相似文献
17.
Weinstein JR Zhang M Kutlubaev M Lee R Bishop C Andersen H Hanisch UK Möller T 《Neurochemical research》2009,34(3):445-452
Microglia are the immune cells of the CNS. Brain injury triggers phenotypic changes in microglia including regulation of surface
antigens. The serine proteinase α-thrombin can induce profound changes in neural cell physiology via cleavage of proteinase-activated
receptors (PARs). We recently demonstrated that pharmaceutical-grade recombinant human α-thrombin (rh-thr) induces a restricted
set of proteolysis-dependent changes in microglia. CD95(Fas) is a cell-death receptor that is up-regulated in microglia by
inflammatory stimuli. Here we characterized the effect of rh-thr on CD95(Fas) expression in the N9 microglial cell line. Dose–response
and time course studies demonstrated maximal effects at 100 U/ml and 24 h, respectively. Regulation of expression was seen
at both the surface protein and steady-state mRNA levels. The rh-thr-induced effects were mimicked by PAR1 agonist peptides and blocked by pharmacologic inhibitors selective for extracellular signal-regulated kinase 1/2 (ERK 1/2).
Rh-thr also induced a rapid and sustained phosphorylation of ERK 1/2. Thrombin-induced regulation of CD95(Fas) could modulate
the neuroinflammatory response in a variety of neurological disorders. 相似文献
18.
Kongphanich A Hieda M Kurokawa K Murata T Kobayashi N 《Biochemical and biophysical research communications》2002,294(3):714-718
In spite of carrying large amount of Fas death receptor on the cell surface, Human T cell lymphotropic virus type-I (HTLV-I)-infected T cell lines are resistant to Fas-mediated cytotoxicity. We investigated the mechanism(s) of HTLV-I-induced Fas resistance. Western blotting and enzymatic activity analyses revealed that the Fas-elicited apoptotic signal in HTLV-I-infected T cells was intervened at the level(s) prior to the activation of caspase-8. Upon stimulation, the clustering of Fas receptors scarcely occurred in HTLV-I-infected cells. Cycloheximide treatment converted the resistant cells to sensitive cells; the presence of short-lived anti-apoptotic molecule(s) that can block the caspase-8 activation within HTLV-I-infected T cells is suggested. 相似文献
19.
Apoptosis in human embryo development: 1. Cerebral cortex 总被引:1,自引:2,他引:1
We investigated the apoptosis at the beginning of human cerebral cortex development, in the 6th week of embryogenesis, Carnegie stages 16 and 17. Attention was focused on the dorsal wall of the telencephalon to the ventricular zone of proliferation and to the postmitotic zone with beginning of neuronal migration. We identified apoptotic cells in tissue sections by propidium iodide staining, TUNEL and immunohistochemistry for Fas(APO-1/CD95). We determined the distribution and the percentage (reported to the propidium iodide stained nuclei) of apoptotic TUNEL-positive and Fas(APO-1/CD95)-positive cells. TUNEL-positive apoptotic cells in the proliferative zone were 20% in stage 16 and 60% in stage 17. TUNEL-positive apoptotic cells in the postmitotic zone were 8% in stage 16 and 30% in stage 17. CD95-positive apoptotic cells in the proliferative zone were 5% in stage 16 and 2% in stage 17. There were no CD95-positive cells in the postmitotic zone. We evidentiated the presence of the suicide receptor Fas(APO-1/CD95) only on a small population of apoptotic neuroblasts in the proliferative zone. The differences between apoptotic distribution and receptors in early corticogenesis suggest that different apoptotic pathways drive the selection of neuronal populations. 相似文献
20.
About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations. 相似文献