Mouse islet cell lysis mediated by interleukin-1-induced Fas

Summary This study was conducted to investigate the possible involvement of Fas in β-cell death in insulitis of Type 1 (insulin-dependent) diabetes mellitus. Although primary cultured Balb/c mouse islet cells did not express Fas mRNA, 4–12 hours of treatment with 10²-10⁵ U/l of mouse interleukin-1 α (IL-1α) induced the expression of Fas mRNA. Surface Fas expression was detected by immunofluorescence flow cytometry using a non-cytolytic anti-Fas monoclonal antibody after 6 or 12 h of incubation with 10³ U/l of IL-1α. Primary islet cells were resistant to an agonistic anti-Fas monoclonal antibody. However, 12 h pretreatment with IL-1α sensitized islet cells to its cytolytic effect. Significant cell death was observed 24 h after the addition of anti-Fas, and progressively increased until 72 h, when specific ⁵¹Cr release was 72 ± 6 %. Agarose gel electrophoresis of DNA extracted from cells exposed to IL-1α and agonistic anti-Fas showed internucleosomal DNA fragmentation, a hallmark of apoptotic cell death. Since the Fas antibody showed no cross-reactive activity of tumour necrosis factor (TNF), the cytotoxic effect was not mediated by TNF receptors. A protein synthesis inhibitor cycloheximide augmented Fas-mediated islet cell death. The Fas-mediated killing of islet cells was not l-arginine-dependent, or blocked by N^G-monomethyl-l-arginine. β-TC1 cells also expressed Fas mRNA when exposed to IL-1α or IL-1α plus interferon-γ. These observations suggest that Fas-mediated apoptosis may be a mechanism of islet cell death in autoimmune insulitis. [Diabetologia (1996) 39: 1306–1312] Received: 5 April 1996 and in revised form: 7 August 1996     

Vascular endothelial cells and smooth muscle cells differ in expression of Fas and Fas ligand and in sensitivity to Fas ligand-induced cell death: implications for vascular disease and therapy

Fas ligand (FasL) is a death factor that induces apoptosis in cells bearing its receptor, Fas. Fas and FasL have been detected in the vessel wall, and it has been proposed that Fas-mediated apoptosis has a role in physiological and pathological cell turnover in the vasculature. Here, we evaluated the expression of Fas in the presence and absence of cytokines on both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). We also examined the sensitivity of ECs and VSMCs to Fas-mediated apoptosis induced by exposure to multiple Fas agonists: soluble FasL, anti-Fas antibody, and membrane-bound FasL resulting from transduction with a replication-defective adenovirus expressing FasL (Adeno-FasL). Cell-surface FasL expression was detected on human ECs with the use of 4 anti-FasL antibodies, whereas cell-surface FasL expression was not detected on VSMCs. Unstimulated ECs expressed relatively low levels of Fas, but expression was upregulated after treatment with tumor necrosis factor-alpha (TNF-alpha) or interferon gamma (IFN-gamma). In contrast, VSMCs expressed relatively high levels of Fas, and treatment with TNF-alpha or IFN-gamma induced little or no upregulation under the conditions of these assays. ECs were resistant to death after exposure to soluble FasL or agonist anti-Fas antibody and also after infection with Adeno-FasL in the presence or absence of cytokine treatment. In contrast, VSMCs remained viable in the presence of soluble FasL or agonist anti-Fas antibody, but they underwent apoptosis after infection with Adeno-FasL. IFN-gamma enhanced Adeno-FasL-induced death of VSMCs, but TNF-alpha did not. These findings provide insights about the potential role of Fas-mediated apoptosis in the vessel wall and suggest strategies to treat proliferative vascular diseases by exploiting the differential sensitivity of ECs and VSMCs to FasL-induced cell death.     

Increased expression of Fas (APO-1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation

Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.     

Expression of Fas-associated death domain-like interleukin-1β-converting enzyme (FLICE) inhibitory protein (FLIP) in human articular chondrocytes: possible contribution to the resistance to Fas-mediated death of in vitro cultured human articular chondrocytes

Although chondrocyte apoptosis has been noted in arthritic joints, the mechanism is not clear. To investigate whether Fas-mediated apoptosis has a role in this process, the presence of Fas mRNA and expression of cell surface Fas protein in monolayer-cultured human articular chondrocytes was analyzed. Fas mRNA was found in all chondrocyte samples analyzed; moreover, the majority of cells in chondrocyte populations expressed cell-surface Fas (12-90%, average 49%). Nevertheless, treatment with an agonistic anti-Fas antibody did not induce significant apoptosis in these chondrocytes in vitro. However, it was also found that chondrocytes express Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), a molecule which blocks Fas-mediated apoptosis. Correspondingly, activation of caspase-8 was minimal in these cultured chondrocytes. In conclusion, although human articular chondrocytes do express cell-surface Fas, this receptor did not fully mediate death-inducing signals in vitro. This resistance to Fas may be partly due to the constitutive expression of FLIP.     

7-Ketocholesterol predisposes human aorta smooth muscle cells to Fas-mediated death

Human vascular smooth muscle cells (HVSMCs) are resistant to Fas-mediated death under normal physiological conditions. However, HVSMC death by activation of the receptor pathway was reported in the atherosclerotic lesions. In this study, we investigated whether 7-ketocholesterol, one of the major cholesterol oxides in the lesions, altered resistance of HVSMC to Fas-mediated death pathway. Cross-linking of Fas receptor with agonistic anti-Fas antibody (CH11) in the presence of 7-ketocholesterol induced death in human aorta smooth muscle cells (HAoSMC) as detected by morphology, viability, and DNA fragmentation. The agonistic anti-Fas antibody, however, did not induce death in the presence of 7alpha-hydroxycholesterol or cholesterol. The HAoSMC death was significantly inhibited by an antagonistic Fas receptor (FasR) antibody and by expression of dominant negative Fas-associated death domain containing protein (DN-FADD) using adenoviruses. Activation of caspase-3 was observed in HAoSMC destined to death. HAoSMC death was significantly inhibited by pharmacological caspase inhibitor, z-VAD and z-DEVD, and baculovirus caspase inhibitor p35. 7-Ketocholesterol impaired mitochondrial transmembrane potential and ATP production. Overexpression of bcl-xL also significantly inhibited HAoSMC death. In dying HAoSMC, bax was translocated from the cytosol to mitochondria and cytochrome c was released from mitochondria into the cytosol. This is the first report demonstrating implication of the oxysterol in Fas-mediated death pathway. The present study proposes that 7-ketocholesterol would contribute to loss of HVSMC in the atherosclerotic lesions by altering resistance to receptor-mediated death pathway.     

Expression and function of Fas (APO-1/CD95) in patient myeloma cells and myeloma cell lines

Cross-linkage of the Fas antigen induces programmed cell death in many normal and malignant lymphoid cells by a process known as apoptosis. In this study, we examined the sensitivity of myeloma cell lines and patient plasma cells to a cytolytic anti-Fas monoclonal antibody (MoAb). Eight of 10 myeloma cell lines were induced to undergo programmed cell death by anti-Fas MoAb as determined by DNA fragmentation and morphologic changes. Of the two myeloma cell lines that were resistant to anti-Fas treatment, one did not express the Fas antigen. Only the U266 cell line expressed Fas, but was not killed by the anti0Fas MoAb. To extend these studies, we have examined the expression and function of Fas in freshly isolated CD38hiCD45neg-int plasma cells from patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and primary amyloidosis (AL). By three-color flow cytometry, we found Fas expression in CD38hiCD45neg-int plasma cells from all patient groups to be variable, as Fas was expressed in 15 of 28 MM, 3 of 6 MGUS, and 2 of 7 AL patients. In morphologic studies of apoptosis, Fas-positive myeloma cells in patient bone marrow mononuclear cell (MNC) cultures appeared to be resistant to anti-Fas-mediated apoptosis. By contrast, purified myeloma cells from the same patient were sensitive to anti-Fas treatment, suggesting the presence of a protective factor(s) in unseparated MNC cultures that may inhibit Fas-induced apoptosis of plasma cells. Of interest, serum from normal individuals and myeloma patients also protected myeloma cell lines from undergoing Fas-mediated apoptosis. These studies show that Fas expression in myeloma cell lines and CD38hiCD45neg-int patient plasma cells is variable and may reflect a variance in the maturation status of the various plasma cell populations. Moreover, Fas-mediated killing of patient cells and myeloma cell lines was also variable, which may be influenced, in part, by the presence of a soluble protective factor.     

Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists

Human immunodeficiency syndrome (HIV) infection leads to a progressive loss of T-cell-mediated immunity associated with T-cell apoptosis. We report here that CD4+ and CD8+ T cells from HIV-1-infected persons are sensitive to Fas (CD95/APO-1)-mediated death induced either by an agonistic anti-Fas antibody or by the physiologic soluble Fas ligand, although showing no sensitivity to tumor necrosis factor alpha-induced death. CD4+ and CD8+ T-cell apoptosis induced by Fas ligation was enhanced by inhibitors of protein synthesis and was prevented either by a soluble Fas receptor decoy or an antagonistic anti-Fas antibody. Fas- mediated apoptosis could also be prevented in a CD4+ or CD8+ T-cell- type manner (1) by several protease antagonists, suggesting the involvement of the interleukin-1beta (IL-1beta)-converting enzyme (ICE)- related cysteine protease in CD4+ T-cell death and of both a CPP32- related cysteine protease and a calpain protease in CD8+ T-cell death; and (2) by three cytokines, IL-2, IL-12, and IL-10, that exerted their effects through a mechanism that required de novo protein synthesis. Finally, T-cell receptor (TCR)-induced apoptosis of CD4+ T cells from HIV-infected persons involved a Fas-mediated death process, whereas TCR stimulation of CD8+ T cells led to a different Fas-independent death process. These findings suggest that Fas-mediated T-cell death is involved in acquired immunodeficiency syndrome (AIDS) pathogenesis and that modulation of Fas-mediated signaling may represent a target for new therapeutic strategies aimed at the prevention of CD4+ T-cell death in AIDS.     

Enhanced Sensitivity to CD95-Induced Apoptosis in ob/ob Mice

Hepatocyte apoptosis was recently described for NASH patients. The pathomechanisms are incompletely understood, but upregulation of the death receptor Fas was detectable on hepatocytes of NASH patients. We analyzed the sensitivity of fatty liver against CD95/Fas-mediated apoptotic cell death by injection of agonistic anti-Fas antibody (Jo2) in obese ob/ob mice and lean control animals. Ob/ob mice died within 12 hrs, whereas control animals survived. Liver enzymes were significantly increased compared to those in control mice (P < 0.001). Histological analysis and also TUNEL assay of liver sections from ob/ob mice exhibited massive liver injury. Activity of caspase 3 was significantly more enhanced in livers of ob/ob mice after Jo2 challenge. The increased sensitivity was confirmed in vitro by using ob/ob-derived primary hepatocytes. CD95 expression was similar in ob/ob and control mice. However, hepatocytes from ob/ob mice revealed a decreased mitochondrial membrane potential, suggesting that mitochondria play a potential role in this increased susceptibility. Juergen Siebler and Markus Schuchmann contributed equally to the work.     

The susceptibility of Philadelphia chromosome positive cells to FAS-mediated apoptosis is not linked to the tyrosine kinase activity of BCR-ABL

We investigated whether inhibition of the BCR-ABL tyrosine kinase by the CGP57418B compound would render chronic myeloid leukaemia (CML) cells susceptible to Fas (CD95, Apo-1)-mediated cell death. Only two (AR230 and SD1) out of 10 BCR-ABL positive cell lines were found to express the CD95 protein. No change in Fas expression was observed in any of the 10 cell lines after 48 h exposure to CGP57418B. AR230 cells were resistant and SD1 cells were partially resistant to Fas-mediated apoptosis induced by ligation of the Fas receptor to an anti-Fas IgM antibody. Pre-incubation with 1 μ M CGP57418B did not change the susceptibility of these cell lines to Fas-mediated cell death. Similar results were observed in experiments with CD34⁺ cells from CML patients and from normal individuals. The data suggest that, in contrast to some cytotoxic drugs, the CGP57148B tyrosine kinase inhibitor utilizes a pathway other than the CD95 system in order to induce apoptosis in CML cells.     

The Fas antigen is detected on immature B cells and the representative cell lines show Fas-mediated apoptosis

The expression and functions of Fas antigen, a major regulator of apoptosis, in T-cell selection have been intensively investigated, but little is known about its expression in immature B cells which are also selected in the bone marrow, and plasma cells which are at the terminal stage of B-cell differentiation and are designed to die. We examined bone marrow cells and found Fas antigen on these cells at low levels. Next, we analysed Fas expression and susceptibility to anti-Fas antibody-mediated apoptosis on B-cell lines representing various stages of differentiation. We also examined the expression of Bcl-2 and Bax on these lines, which were intimately related to apoptosis. Fas antigen was almost negative on pre-pre-B cell lines and was detected on pre-B-cell lines at low levels. All plasma cell lines expressed Fas at a low to moderate level. Some cell lines with peripheral B-cell phenotype expressed Fas antigen. Except for the Burkitt cell lines and one plasma cell line, susceptibility to Fas-mediated apoptosis depended on Fas expression. Bcl-2 protein was detected on all but one cell line and Bax was detected on 15/23 lines, but neither were related to cellular differentiation or Fas expression.     

Involvement of caspase-2 long isoform in Fas-mediated cell death of human leukemic cells

Engagement of the plasma membrane receptor Fas can induce apoptosis of leukemic cells. Signaling through Fas requires the formation of a death-inducing signaling complex (DISC) that involves the cytoplasmic domain of Fas, the adaptor molecule FADD/MORT-1, and procaspase-8. The present study investigated whether another caspase, known as procaspase-2L, played a role in Fas-mediated cell death. A series of human leukemic variant cells was derived by stable transfection with a CASP2L antisense construct (CASP2L/AS). Specific down-regulation of procaspase-2L decreased the sensitivity of these cells to apoptosis induced by an agonistic anti-Fas antibody (Ab, clone CH11), as determined by studying DNA fragmentation, chromatin condensation, and externalization of phosphatidylserine on the plasma membrane. In leukemic cells transfected with an empty vector, anti-Fas Ab treatment activated caspase-8, decreased the expression of the BH3 domain-only protein Bid, triggered the release of cytochrome c from the mitochondria to the cytosol, and activated caspase-3. All these events could not be observed when CASP2L/AS cells were similarly treated with anti-Fas Abs. CASP2L/AS transfection did not inhibit the formation of the DISC and no direct interaction between procaspase-2L and either Fas or FADD or procaspase-8 was identified. Down-regulation of procaspase-2L inhibited anti-Fas Ab-mediated cleavage of c-FLIP (FLICE-inhibitory protein), a protein that interferes with the formation of a functional DISC. These results suggest that the long isoform of caspase-2 plays a role in the Fas-mediated pathway to cell death by contributing to caspase-8 activation at the DISC level.     

Expression of Fas/Apo-1 (CD95) and apoptosis in tumor cells from patients with plasma cell disorders

Fas/Apo-1 antigen (CD95) is a cell surface molecule that directly mediates apoptosis. Fas expression was studied in five plasma cell lines, 11 multiple myeloma cases, and three plasma cell leukemia (PCL) cases. Induction of apoptosis by anti-Fas antibody was studied in five plasma cell lines and fresh plasma cells from eight patients. Apoptosis was confirmed by morphologic analysis alone or in combination with DNA electrophoresis analysis. Four of the five cell lines showed Fas expression, three of which showed induction of apoptosis by anti-Fas antibody. One cell line, RPMI 8226, showed the highest sensitivity for Fas-mediated apoptosis. High bcl-2 expression was found in KMS12PE, which showed resistance to Fas-mediated apoptosis despite its Fas expression. Plasma cells from seven fresh cases, including all five cases with high serum lactate dehydrogenase (LDH), showed expression of Fas antigen. Fas-induced apoptosis was found in five cases at various levels, although significant induction of apoptosis was found in only one case. Interestingly, Fas-independent apoptosis was induced during culture without anti-Fas antibody in cases with high serum LDH. These results indicate that plasma cells from aggressive myeloma with high LDH express Fas antigen and undergo apoptosis through either Fas- mediated or Fas-independent pathways. An understanding of the mechanism of apoptosis in malignant plasma cells should contribute to investigations of the pathophysiology of and therapy for myeloma/PCL.     

Fas-associated phosphatase-1 promotes Fas-mediated apoptosis in human colon cancer cells: Novel function of FAP-1

Background and Aim: Fas‐associated phosphatase‐1 (FAP‐1) has been thought as an inhibitor in Fas‐mediated apoptosis. Here, we investigated the role of FAP‐1 in Fas‐mediated apoptosis of human colon cancer cells. Method: The viability of four colon cancer cell lines treated with agonistic anti‐Fas antibody was determined using WST‐1 assay and cell death detection ELISA. pRc/CMV‐FAP‐1 was transfected to a FAP‐1‐negative, Fas‐resistant colon cancer cell line SW480 by lipofection and the clones expressing FAP‐1 protein were selected by limiting dilution. In the clones, expression of 550 genes was analyzed by cDNA microarrays. Protein expression of FAP‐1 and molecules related to apoptosis was examined by western blot. Results: We obtained two FAP‐1 overexpressed clones which were much more susceptible to Fas‐mediated apoptosis than control cells. In the clones, caspase 8 and caspase 3 were fully activated by agonistic anti‐Fas antibody treatment. Bcl‐2 family proteins were not related to the high susceptibility of these clones, because caspase 9 was not activated. Transfection of FAP‐1 did not suppress the survival actions of insulin‐like growth factor (IGF‐1) which enhanced survival signal through Akt phosphorylation. Upregulation in 21 genes and downregulation in 29 genes was revealed by cDNA arrays. We confirmed protein expression of p21 and phosphorylated p21 were much more enhanced in the clones than in control cells. Conclusions: Overexpression of FAP‐1 enhanced susceptibility to Fas‐mediated apoptosis in SW480 and upregulation of p21 may contribute to this phenomenon. Our results indicate a novel function of FAP‐1 in Fas‐mediated apoptosis of human colon cancer cells.     

CD4+ T-cell induction of Fas-mediated apoptosis in Burkitt's lymphoma B cells

Cytotoxic function of CD4+ Th1 cells is mediated by Fas (CD95, APO-1) and its ligand (Fas ligand). Recent studies using nontransformed B cells and the Ramos Burkitt's lymphoma (BL) B-cell line cells show that CD40 ligation at the B-cell surface by activated, CD40 ligand (CD40L)- bearing, CD4+ T cells upregulates Fas expression on B cells and primes B cells for Fas-mediated death signals. In this work, we examine whether this CD4+ T-cell-dependent molecular pathway for Fas upregulation and B-cell apoptosis reflects a peculiarity of the Ramos B- cell line or is applicable to other Burkitt's tumors as well. In 5 of the 6 Epstein-Barr virus-negative BL cell lines examined, the cells constitutively express undetectable or low levels of Fas and are resistant to Fas-mediated signals induced by monoclonal anti-Fas antibody. All 6 of the BL cell line B cells upregulate Fas in response to CD40 ligation, and in 4 of the cases they become sensitive to Fas- mediated death signals. In one BL cell line, the cells are constitutively sensitive to Fas-mediated cytolysis and are unaffected by CD40 signals. Next, we applied these immunologic manipulations to cells from a refractory clinical sample and observed that the tumor cells could be induced to express Fas and undergo apoptosis in our system. These results establish CD4+ T cells and the Fas-Fas ligand system as important immune regulators of Burkitt's lymphoma B cells and indicate that the susceptibility of tumor cells to Fas-mediated death signals can be modulated by specific activation events at the cell surface.     

Oxidized low density lipoprotein potentiation of Fas-induced apoptosis through lectin-like oxidized-low density lipoprotein receptor-1 in human umbilical vascular endothelial cells.

Under normal conditions, vascular endothelial cells are resistant to Fas-mediated apoptosis, although they express detectable Fas on their cell surface. Because oxidized Low density lipoprotein (Ox-LDL) is thought to promote atherogenesis, the potential role that Ox-LDL may play in Fas-mediated apoptosis was investigated in human umbilical vascular endothelial cells (HUVECs), focusing particularly on the involvement of the lectin-like Ox-LDL receptor-1 (LOX-1). HUVECs were treated with agonistic anti-Fas antibody (CH11) and Ox-LDL and then the degree of apoptosis was determined by cell death ELISA. Ox-LDL concentration-dependently sensitized Fas-mediated apoptosis. Flow cytometry demonstrated that Ox-LDL dose-dependently up-regulated cell surface Fas expression. On the other hand, treating HUVECs with Ox-LDL did not lead to any significant change in the expression of death mediators, including Fas, Fas ligand (FasL), FADD, and FLICE as assessed by multiplex polymerase chain reaction amplification. More importantly, these effects of Ox-LDL on Fas-mediated apoptosis were significantly blocked by a neutralizing LOX-1 monoclonal antibody, which can block LOX-1-mediated cellular uptake of Ox-LDL. Ox-LDL may be an important factor involved in the regulation of Fas-induced apoptosis via Ox-LDL/LOX-1 interaction in vascular endothelial cells. The results may provide insights into the pathogenesis of accelerated atherosclerosis in patients with hyperlipidemia.     

Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or gamma radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or gamma radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.     

Shedding of Fas ectodomain that affects apoptosis of hepatocytes occurring in regenerative liver

Background: On the basis of comprehending several membrane proteins undergoing ectodomain shedding, including tumor necrosis factor (TNF) α receptors, we want to know if Fas (CD95/APO-1), which belongs to the TNF receptor (TNFR) superfamily and transduces signals resulting in apoptosis upon binding of Fas ligand (L) or agonistic anti-Fas antibody, is shed on its ectodomain during liver regeneration Methods: After purification of the total membrane protein of hepatocytes, we analyzed Fas ectodomain shedding, using Western blotting, and determined the effect of Fas shedding on the apoptosis of hepatocytes by a statistical method relevant to apoptosis. Results: Fas protein ectodomain shedding occurred at 2, 12, and 36 h after partial hepatectomy, and its level decreased at 2 months after partial hepatectomy. The same results were gained from in-vitro cultured hepatocytes induced by serum from rats after partial hepatectomy. The sensivitity of hepatocytes to agonistic anti-Fas antibody increased significantly after they were treated with serum from rats after partial hepatectomy. However, the expression of Fas mRNA and Fas protein did not affect the liver regeneration. Conclusions: Fas ectodomain shedding might be an important mechanism in controlling hepatocyte apoptosis during liver regeneration. Received: February 25, 2002 / Accepted: May 31, 2002     

Inhibition of mouse hepatocyte apoptosis via anti-Fas ribozyme

AIM: To investigate the effects of anti-Fas ribozyme on Fas expression and apoptosis in primary cultured mouse hepatocytes. METHODS: Mouse hepatocytes were isolated by using collagenase irrigation. A hammerhead ribozyme targeting the Fas mRNA was constructed, and transfected into mouse hepatocytes via Effectene. Then Fas expression in mouse hepatocytes was detected by RT-PCR and western blotting. After being treated with anti-Fas antibody (JO2), hepatocytes viability was measured with MTT assay. Caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to Annexin V-FITC apoptosis detection kit. RESULTS: Fas expressed in primary mouse hepatocytes. Fas expression in hepatocytes transfected with anti-Fas ribozyme was decreased remarkably and correlated with the resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. CONCLUSION: Anti-Fas ribozyme can remarkably decrease the Fas expression in mouse hepatocytes, thus inhibit Fas-mediated apoptosis in hepatocytes. It is suggested that anti-Fas ribozyme could significantly increase the resistance of transplanted hepatocytes to apoptosis and improve the survival of transplanted hepatocytes.