Apoptosis of murine cultured biliary epithelial cells induced by glycochenodeoxycholic acid involves Fas receptor and its ligand

Fas-mediated apoptosis of biliary epithelial cells (BECs) is suggested as a main effector process in immune-mediated biliary diseases. Glycochenodeoxycholic acid (GCDCA), a conjugated hydrophobic bile acid, is known to cause apoptosis of hepatocytes via the direct activation of Fas receptor (FasR). In this study, we investigated the apoptotic effect of GCDCA on cultured murine intrahepatic BECs. It was found that GCDCA induced apoptosis of BECs derived from BALB/c mice in a dose- and incubation time-dependent manner. The morphology and TUNEL positivity for the apoptotic process induced by GCDCA were similar to those induced by beauvericin (BV) which is known to cause apoptosis by a direct activation of caspase-3, a member of the caspase family, at its downstream. The GCDCA-induced apoptosis of BECs was accompanied by an up-regulation of FasR, Fas ligand (FasL), and also caspase-3 expression. GCDCA did not induce the apoptosis of BECs derived from C3H · MRL-Fas^lpr mice possessing a non-functioning FasR, supporting that the GCDCA-induced apoptosis of BECs in BALB/c involves the Fas system. Furthermore, GCDCA induced an increase in interleukin-18 (IL-18) mRNA in BECs and secretion of activated IL-18 from BECs of BALB/c, which might have led to an up-regulation of FasL mRNA and protein expression in BECs followed by FasR/FasL interaction. These results suggest that GCDCA induces apoptosis of BECs through FasR/FasL interaction via an autocrine/paracrine effect, IL-18 is responsible for the expression of FasL in BEC, and the up-regulation of caspase-3 expression is involved in this model. This model could be useful for molecular and genetic studies of Fas-mediated apoptosis of BECs.     

Regulation of inducible nitric oxide synthase expression in bovine ovarian granulosa cells

Nitric oxide (NO) is a potential regulator of ovarian follicle growth, and ovarian granulosa cells reportedly generate NO in response to gonadotrophins, suggesting that the regulated form of nitric oxide synthase (iNOS) is present. The objectives of the present study were to gain insight into the expression and role of iNOS in the follicle. Messenger RNA encoding iNOS was detected in granulosa cells, and abundance was higher in growing dominant follicles compared to subordinate follicles (P<0.01). FSH (P<0.05) and IGF1 (P<0.01) stimulated oestradiol secretion and iNOS mRNA abundance in granulosa cells in vitro, whereas FGF2 (P<0.05) and EGF (P<0.01) decreased oestradiol secretion and iNOS expression. The addition of an anti-oestrogen prevented FSH-induced iNOS mRNA accumulation. Inhibition of endogenous NO production did not affect steroidogenesis in granulosa cells, but increased FasL mRNA abundance, caspase-3 activation and the incidence of apoptotic cell death (P<0.05). These results demonstrate that iNOS is expressed in ruminant granulosa cells and is regulated by gonadotrophins and oestradiol. Physiological levels of NO may contribute to the survival of granulosa cells.     

Fas ligand is highly expressed in acute leukemia and during the transformation of chronic myeloid leukemia to blast crisis.

Fas ligand (FasL) induces apoptosis in susceptible Fas-bearing cells and is critically involved in regulating T-cell immune responses. It is highly expressed in several human malignancies, and a role in the suppression of antitumor immune responses has been suggested. We evaluated FasL expression in leukemia and normal hematopoietic cells. By Western blotting, all acute leukemic cell lines (n = 9) and primary samples of acute leukemic marrow (n = 4) revealed high levels of FasL. In contrast, much weaker signals were observed in samples of normal marrow (n = 5), and either weak or intermediate expression was seen in chronic myeloid leukemia (CML) in chronic phase (n = 7). Additional leukemic samples were examined by immunohistochemistry. Staining for FasL was negative in 7 of 9 cases of chronic-phase CML, whereas all cases of CML in blast crisis (n = 6), acute lymphoblastic leukemia (n = 6), and acute myeloid leukemia (n = 11) stained strongly in 60 to 100% of nucleated cells. FasL+ leukemic cell lines did not trigger Fas-mediated apoptosis in either Jurkat cells or activated human T lymphocytes, possibly related to the intracellular location of the ligand. Western analysis of normal marrow subpopulations revealed that most FasL in marrow mononuclear cells was expressed by CD7+ lymphocytes. FasL also was strongly expressed in CD34+ hematopoietic progenitor cells from both normal and chronic-phase CML marrow, suggesting a correlation with primitive maturation stage. In summary, high levels of FasL expression were associated with aggressive biologic behavior in leukemia, including transformation of CML to blast crisis. This could potentially represent a response to loss of proapoptotic Fas signaling, which is known to occur in acute leukemic blasts.     

Cholangiocarcinomas express Fas ligand and disable the Fas receptor

Cholangiocarcinoma is a highly-malignant adenocarcinoma originating from cholangiocytes. Current concepts support escape from immune surveillance using aberrant expression of Fas ligand (FasL) and dysregulation of receptor (FasR) signaling as a potential mechanism for tumor progression. Our aims were to determine if altered expression of FasR and FasL or changes in expression of FLICE inhibitor (I-FLICE) allow cholangiocarcinoma cells to escape immune surveillance. Human cholangiocarcinoma cell lines were evaluated for the functional expression of FasR and FasL by (1) quantitating apoptosis after incubation of cells with agonistic antibodies and (2) an in vitro cell death assay involving coculture of cholangiocarcinoma cells with Fas-sensitive thymocytes. I-FLICE antisense treatment was performed by stable transfection with complementary DNA (cDNA) for I-FLICE in the reverse orientation. We found that normal cholangiocytes in vivo express FasL. Human cholangiocarcinoma cell lines express both FasL and FasR and I-FLICE. FasL expressed by cholangiocarcinomas in vitro induced lymphocyte cell death (70% after 24 hours). Despite the expression of FasR, exposure of the cells to agonistic antibodies (500 ng/mL) induced only minimal apoptosis in the Jurkat cells. Antisense treatment of cholangiocarcinomas in vitro with I-FLICE reduced protein expression of I-FLICE by 90% to 95% and increased Fas-mediated apoptosis 2-fold. We concluded that cholangiocarcinomas escape immune surveillance either by disabling FasR signaling through the expression of I-FLICE and/or increased FasL expression to induce apoptosis of invading T cells. Reduction of I-FLICE expression in cholangiocarcinoma cells restored Fas-mediated apoptosis. Therapeutic maneuvers to inhibit expression of I-FLICE may aid in the treatment of cholangiocarcinoma.     

Stem cell factor increases the expression of FLIP that inhibits IFNgamma -induced apoptosis in human erythroid progenitor cells

Interferon gamma (IFNgamma) acts on human erythroid colony-forming cells (ECFCs) to up-regulate Fas, without a demonstrable change of Fas ligand (FasL) or Fas-associated DD-containing protein (FADD) expression and activates caspase-8 plus caspase-3, which produce apoptosis. Our previous data showed that stem cell factor (SCF) reduced the inhibitory effect of IFNgamma on human ECFCs when both factors were present in the cultures. However, the mechanism by which SCF prevents IFNgamma-induced apoptosis in ECFCs is unclear. In this study we used highly purified human ECFCs to investigate the mechanism of the effect of SCF on IFNgamma-induced apoptosis. Because the binding of FasL to Fas is the first step of the apoptosis cascade and IFNgamma strongly up-regulates Fas expression, we added FasL (50 ng/mL) to the cultures with IFNgamma to accentuate the IFNgamma-induced activation of caspase-8 and caspase-3 plus subsequent apoptosis. SCF (100 ng/mL) clearly inhibited the activation of caspase-8 and caspase-3 induced by IFNgamma and/or FasL, and it also reduced apoptosis as measured by the terminal dUTP nick-end labeling (TUNEL) assay. SCF did not decrease the surface expression of Fas on the ECFCs. FADD-like interleukin 1 beta (IL-1beta)-converting enzyme (FLICE)-inhibitory protein (FLIP) has been reported to interact with FADD and/or caspase-8 at the death-inducing signaling complex (DISC) level following Fas stimulation and acts as a dominant-negative caspase-8. SCF increased FLIP mRNA and protein expression, concomitant with reduced apoptosis, whereas IFNgamma and/or FasL did not change FLIP expression. Reduction of FLIP expression with antisense oligonucleotides decreased the capacity of SCF to inhibit IFNgamma-induced apoptosis, demonstrating a definite role for FLIP in the SCF-induced protection of ECFCs from IFNgamma-initiated apoptosis.     

Fluvastatin upregulates inducible nitric oxide synthase expression in cytokine-stimulated vascular smooth muscle cells

Nitric oxide (NO) production by inducible NO synthase (iNOS) may play an important role in the pathogenesis of atherosclerosis. Although fluvastatin has been shown to reduce progression of atherosclerosis, it is not known whether it regulates iNOS expression. We investigated the effects of fluvastatin on iNOS expression and subsequent NO synthesis in vascular smooth muscle cells (VSMCs) and the mechanism by which fluvastatin exerts its effects. Fluvastatin significantly increased interleukin-1ss (IL-1ss)-induced nitrite production by VSMCs in a time-dependent (0 to 24 hours) and dose-dependent (10(-)(8) to 10(-)(5) mol/L) manner. Increased nitrite production by fluvastatin was accompanied by increased iNOS mRNA and protein accumulation. IL-1ss induced nuclear factor-kappaB activation in VSMCs, which was not affected by fluvastatin. Exogenous mevalonate significantly prevented the stimulatory effect of fluvastatin on nitrite production. Cotreatment with geranylgeranyl-pyrophosphate also reversed the effect of fluvastatin. Furthermore, both Rho inhibitor C3 exoenzyme and Rho kinase inhibitor Y-27632 significantly increased IL-1ss-induced nitrite accumulation in VSMCs. These results demonstrated that fluvastatin upregulates iNOS expression and subsequent NO formation in rat VSMCs through inhibition of Rho.     

Expression of apoptosis-related proteins in rat with induced colitis

Background and aims Inflammatory bowel diseases (IBD) evoke a damage-repair process accompanied by the activation of apoptotic genes. Data on transglutaminase (TG) expression in apoptotic cells in inflamed colonic epithelium has not been reported, although TG cross-links proteins within typical apoptotic bodies in various cell lines. In an experimental model of colitis we investigated the expression of different markers of apoptosis related to the degree and development of colonic inflammation.Methods Two studies were performed: (a) Colitis was induced by the administration of 2,4,6-trinitrobenzen sulfonic acid (TNBS) at a dose of 10 or 20 mg per rat in 50% ethanol, and the rats were killed 1 week later; (b) Colitis was induced by 20 mg TNBS and the rats were killed 3 days, 1, 2, and 4 weeks thereafter. The colon of rats was macroscopically assessed, and biopsies were histologically assessed and immunoprobed for FasL, FasR, p53 and tTG. Cell death was detected by TUNEL, and TG activity was assayed on colon homogenates.Results Study A: According to enhanced TUNEL positivity, FasR/FasL and p53 expression increased depending on the severity of the colitis. Study B showed increased p53 expression at day 3 while FasR/FasL coexpression peaked at 1 week. In both studies tTG was mainly expressed in the extracellular matrix of damaged tissue and in the submucosa.Conclusions Our findings suggest that expression of apoptosis markers is related to the degree of colitis and show that apoptosis is sustained by both p53 and FasR/FasL pathways, depending on the phase of colitis development. Moreover, the lack of TG staining in typical apoptotic bodies may account for a perturbation of the cross-linked apoptotic envelope that may be an important determinant in the development of immune response in ulcerative colitis.     

Tumour necrosis factor-alpha and nitric oxide mediate apoptosis by D-galactosamine in a primary culture of rat hepatocytes: exacerbation of cell death by cocultured Kupffer cells.

BACKGROUND: Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. OBJECTIVES: Whether PGE1 protects against D-galactosamine (D-GalN)-associated hepatocyte cell death by the regulation of tumour necrosis factor-alpha (TNF-alpha) and/or nitric oxide (NO) in hepatocytes or cocultured Kupffer cells was examined. METHODS: Anti-TNF-alpha antibodies were used to evaluate the role of TNF-alpha during D-GalN cytotoxicity and its protection by PGE1 in cocultured hepatocytes and Kupffer cells. Cell apoptosis and necrosis were assessed by DNA fragmentation and lactate dehydrogenase release, respectively. Nitrite+nitrate (NOx), as NO end products, and TNF-alpha concentrations were measured in the culture medium. The role of NO was determined by measuring inducible NO synthase (iNOS) expression and the effect of its inhibition during d-GalN cytotoxicity and its protection by PGE1. RESULTS: D-GalN enhanced hepatocyte cell death associated with high TNF-alpha and NOx levels in a culture medium. Anti-TNF-alpha and iNOS inhibition suggested that TNF-alpha was mediating apoptosis, but not necrosis, through the stimulation of NO production. The antiapoptotic activity of PGE1 was associated with a reduction of NO production, but was blocked by iNOS inhibition. This apparent contradiction was explained by the ability of PGE1 to enhance iNOS expression shortly after its administration and inhibit it later during d-GalN treatment. Anti-TNF-alpha antibodies did not reduce the exacerbation of d-GalN-associated cell death in hepatocytes by cocultured Kupffer cells. CONCLUSION: TNF-alpha mediates D-GalN-induced apoptosis via NO production in cultured hepatocytes. The protective effect of PGE1 against D-GalN-induced apoptosis is probably through the induction of low iNOS expression that was followed by a reduction of iNOS expression and NO production induced by the hepatotoxin. The exacerbation of hepatocyte cell death by Kupffer cells was not related to TNF-alpha and NO.     

Cross-Talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia

Recent studies have shown the involvement of Fas/Fas ligand (FasL) system and nitric oxide (NO) in ovarian follicle atresia. Here we asked whether Fas/Fas ligand system interacts with NO using rat granulosa cell culture. Soluble recombinant Fas ligand (rFasL), at 100 ng/ml, significantly decreased cell viability, as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, in the presence of 200 U/ml interferon-gamma, whereas the concurrent addition of a caspase inhibitor, Z-VAD-FMK, at 20 microm, significantly inhibited rFasL-induced cytotoxicity. Hoechst 33342 staining and flow cytometric analysis confirmed the induction of apoptosis in granulosa cells by 100 ng/ml rFasL in the presence of interferon-gamma, which was blocked by the concomitant addition of an NO donor, S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated that rFasL significantly up-regulated caspase-3, -8, and -9 activities in granulosa cells, which were attenuated by concurrent treatment with S-nitroso-N-acetylpenicillamine. Real-time quantitative RT-PCR revealed a significant decrease in inducible NO synthase mRNA levels in rFasL-induced apoptotic granulosa cells. In conclusion, we demonstrated the involvement of Fas/FasL system in inducing apoptosis through activation of a caspase-mediated cascade in rat granulosa cells, which is coupled with a decrease in inducible NO synthase expression. We further showed that NO inhibited Fas/FasL system-induced apoptosis by suppressing activation of the caspases, pointing to a cross-talk between Fas/FasL system-induced apoptosis pathway and NO-mediated antiapoptotic pathway in ovarian follicle atresia.     

一氧化氮可能参与多巴胺对PC12细胞凋亡的诱导作用

目的：探讨多巴胺（DA）诱导的PC12细胞凋亡是否与内源性一氧化氮（NO）生成有关。方法：酶还原法测定NO浓度，RT－PCR法检测诱导型一氧化氮合酶（iNOS)和caspase-3 mRNA表达水平，免疫细胞化学方法和蛋白印迹法分别检测活化型caspase-3蛋白和iNOS蛋白水平。结果：在多巴胺诱导PC12细胞凋亡过程中，iNOS mRNA和蛋白表达明显增加，内源性NO生成增多，caspawse-3mRNA和活化型caspase-3蛋白的表达也明显增加，结论：内源性NO可能参与多巴胺对PC12细胞凋亡的诱导作用。     

Protective effect of spironolactone on endothelial cell apoptosis

Human umbilical vein endothelial cells (HUVECs) undergo apoptosis in response to serum deprivation. We show that the nonspecific mineralocorticoid receptor antagonist, spironolactone, protects from caspase-3 activation induced by serum deprivation in contrast to the selective mineralocorticoid receptor antagonist, eplerenone, that is nonprotective. We also demonstrate that progesterone, hydrocortisone, and dexamethasone all protect HUVECs from serum-deprivation-induced caspase-3 activation, whereas aldosterone and dihydrotestosterone have no effect. Spironolactone has been demonstrated to display agonist activity only to the progesterone receptor (PR), and we additionally show that spironolactone and progesterone, but not eplerenone, inhibit mitochondrial cytochrome c release and cleavage of nuclear poly (ADP-ribose) polymerase (PARP) and increase cell viability. Additionally, the PR antagonist mifepristone (RU486) partially blocked the inhibitory effect of both spironolactone and progesterone on caspase-3 activation, cytochrome c release, and nuclear PARP cleavage. Nitric oxide (NO) protects HUVECs from apoptosis in response to various stimuli including serum-deprivation; however, the NO synthase inhibitor N-monomethyl-l-arginine, did not abolish inhibition of caspase-3 activation or PARP cleavage by spironolactone. Thus, we demonstrate that spironolactone protects HUVECs from serum-deprivation-induced apoptosis by inhibition of caspase-3 activity, cytochrome c release and PARP cleavage by a NO-independent mechanism; further, this effect is likely mediated by the agonist properties of spironolactone toward the PR.     

Activation of caspase-8 in transforming growth factor-beta-induced apoptosis of human hepatoma cells.

Transforming growth factor-beta1 (TGF-beta1) has been shown to induce apoptosis in normal or transformed hepatocytes. To elucidate the biochemical pathways leading to apoptosis induced by TGF-beta1 in human hepatoma cells (HuH-7), we examined the expression of Bcl-2-related proteins and X-chromosome-linked inhibitor of apoptosis (XIAP), and activation of the caspase cascade following TGF-beta1 treatment. Bcl-xL expression began to decline at 12 hours after TGF-beta1 treatment and progressively decreased to very low levels in a time-dependent manner. Bax expression showed a little change throughout the experiment. On the other hand, activation of caspase-8 was clearly observed at 36 hours after TGF-beta1 treatment, followed by activation of caspase-9, and caspase-3 was activated at 48 hours after treatment at which time apoptosis of HuH-7 cells was observed. TGF-beta1 significantly decreased XIAP expression in HuH-7 cells. Addition of an inhibitor of caspase-8 or caspase-3 (IETD-FMK or DEVD-CHO) markedly inhibited TGF-beta1-induced apoptosis of HuH-7 cells. Fas/Fas ligand (FasL) interactions in HuH-7 cells were not involved in the apoptotic process. Furthermore, epidermal growth factor (EGF) also completely inhibited TGF-beta1-induced apoptosis of HuH-7 cells by inhibiting activation of the caspase cascade. Our results suggested that activation of caspase-3 initiated through caspase-8 activation is involved in the apoptotic process induced by TGF-beta1 in HuH-7 cells. Our results also showed that down-regulation of the expression of Bcl-xL and XIAP by TGF-beta1 may facilitate activation of caspase-3 in these cells.     

Fas receptor counterattack against tumor-infiltrating lymphocytes in vivo as a mechanism of immune escape in gastric carcinoma

We investigated the presence and functional status of surface expression of the Fas receptor (FasR) and its ligand (FasL) in tumor and tumor-infiltrating lymphocytes (TIL) in gastric carcinoma (n=36) from the primary locus, metastatic gastric carcinoma (n=30) from malignant ascites, and benign gastric mucosa (n=30) for the control. The quantitative analysis was based on the percentage of positive cells by a flow cytometry. The results showed that the membrane-bound FasL molecule was constitutively expressed in primary and metastatic gastric carcinomas as well as normal gastric epithelium in nearly all the patients. In particular, metastatic carcinoma proved to aberrantly express the FasL molecule. On the other hand, FasR expression ranged from minimal or absent in primary and metastatic gastric carcinomas, suggesting that the carcinoma might be rendered less sensitive toward FasR-induced killing. Apoptotic tumor cells detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick and labeling (TUNEL) were barely identified in primary and metastatic carcinomas. In the analysis of TIL, the expression of FasR and FasL, and apoptotic TIL could not usually be observed in primary gastric carcinoma. In metastatic carcinoma, however, there was significant overexpression of FasR and FasL in immune TIL associated with a higher frequency of apoptotic cell death detected by TUNEL. The results suggest that metastatic carcinoma expressing FasL, but not FasL⁺ primary carcinoma, might evade the immune attack by apoptotic depletion of activated TIL through the FasR/FasL systems. These results provide the direct and quantitative evidence of FasR counterattacks and/or paracrine fratricides as a mechanism of tumor-immune escape in vivo in human cancer. Received: 29 March 2000 / Accepted: 30 June 2000     

Dup-697, a specific COX-2 inhibitor,suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation

This investigation was designed to assess the effect of DuP-697 on growth and apoptosis in a human chronic myeloid leukemia (CML) cell line (K562 cells) and primary CML cells from CML patient bone marrow. DuP-697 significantly suppressed K562 cells and primary CML cells growth and induced apoptosis in a concentration-dependent manner and the growth-inhibiting effect was independent on Philadelphia chromosome. The IC50 of DuP-697 at 36 h was 31.7 μM. It arrested G1-S phase transmit on cell cycle and its apoptosis activity was partially abrogated by pretreating K562 cells with Z-IETD-fmk, a specific inhibitor of caspase-8. This study suggested that Dup-697 suppresses growth and induces apoptosis on K562 leukemia cells by cell-cycle arrest and caspase-8 activation.     

Interaction between pro-inflammatory and anti-inflammatory cytokines in insulin-producing cells

Pro-inflammatory cytokines cause beta-cell dysfunction and death. The aim of this study was to investigate the interactions between different pro- and anti-inflammatory cytokines and their effects on apoptotic beta-cell death pathways. Insulin-producing RINm5F cells were exposed to different combinations of cytokines. Gene expression analyses of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) were performed by real-time RT-PCR. Cell viability was measured by the MTT assay, NFkappaB activation using a SEAP reporter gene assay, protein expression by western blotting and caspase-3 activity using the DEVD cleavage method. IL-1beta, tumour necrosis factor alpha (TNFalpha) and a combination of all three pro-inflammatory cytokines increased while IFNgamma alone did not affect NFkappaB activity and iNOS gene and protein expression. Interestingly, the anti-inflammatory cytokines IL-4, IL-13 and IL-10 decreased IL-1beta-stimulated NFkappaB activation and iNOS expression. IL-1beta, TNFalpha and the pro-inflammatory cytokine combination also increased MnSOD gene and protein expression. But IL-4, IL-13 and IL-10 did not affect MnSOD expression and did not modulate IL-1beta-stimulated MnSOD expression. Caspase-3 activity was increased by IL-1beta and the pro-inflammatory cytokine combination, and to a lesser extent by TNFalpha. In contrast, IFNgamma had no effect on caspase-3 activity. IL-4, IL-13 and IL-10 decreased caspase-3 activity and increased viability of insulin-producing cells treated with pro-inflammatory cytokines. The anti-inflammatory cytokines counteracted the cytotoxic effects of pro-inflammatory cytokines in insulin-producing cells. This was achieved through the reduction of nitrosative stress. Thus, a balance between the anti-inflammatory and the pro-inflammatory cytokines is of crucial importance for the prevention of pancreatic beta-cell destruction.