Elimination of lymphocytes, but not eosinophils, by Fas-mediated apoptosis in murine schistosomiasis.

Infection with the helminth parasite Schistosoma mansoni is associated with a pathogenic granulomatous response to parasite eggs. Multiple cell types constitute the granuloma with eosinophils achieving numerical dominance. We hypothesize that eosinophil dominance is achieved by selective apoptosis in lymphocytes. We report here that lymphocytes from both the spleens and granulomas of S. mansoni-infected mice undergo apoptosis. We also show that granuloma lymphocytes are more susceptible to Fas-FasL-mediated apoptosis than spleen lymphocytes and this apoptosis may be related to antigen concentration. Conversely, eosinophils from the granuloma and spleens of S. mansoni-infected mice are resistant to apoptosis in vivo and are protected in vitro from Fas-FasL-mediated apoptosis by the absence of FasL expression in the presence of Fas expression. Finally, the apoptotic regulatory molecules Bcl-2, Bcl-xL, and Bax, do not appear to play a significant role in the regulation of eosinophil apoptosis in the schistosome granuloma.     

Resistance to Fas-mediated apoptosis in human lung fibroblast.

The current authors have demonstrated previously that epithelial cell apoptosis, induced by the Fas-Fas ligand pathway, might be involved in fibrosing lung diseases. Whereas lung epithelial cells are sensitive to the Fas-mediated apoptosis, lung fibroblasts may be resistant to Fas-mediated apoptosis and replace damaged epithelial cells. The WI-38 lung fibroblast cell line and primary lung fibroblasts were used to examine the resistant to Fas-mediated apoptosis and the association of anti-apoptotic proteins with this resistance. The administration of agonistic anti-Fas antibody (CH-11) or cycloheximide alone did not induce apoptosis, whereas the co-administration of CH-11 with cycloheximide induced apoptosis in WI-38 cells, in which caspase-8 and -3, but not -9, were activated, and X chromosome-linked inhibitor of apoptosis (ILP) and FLICE-like inhibitor protein (FLIP(L)), but not bcl-xL and bcl-2, were remarkably down regulated. Primary lung fibroblasts were also resistant to Fas-mediated apoptosis, and ILP and FLIP appeared to be involved in this resistance. Furthermore, the results of immunohistochemistry demonstrated that fibroblasts expressed ILP and FLIP(L) proteins in lung tissues from patients with idiopathic pulmonary fibrosis. These results suggest that anti-apoptotic proteins such as X chromosome-linked inhibitor of apoptosis and FLICE-like inhibitor protein may play an important role in preventing Fas-mediated apoptosis in lung fibroblasts, and participate in the development of pulmonary fibrosis.     

Doxorubicin induces Fas-mediated apoptosis in human thyroid carcinoma cells.

Doxorubicin remains the most extensively used drug in the chemotherapy of thyroid cancer. However, drug resistance often limits the efficacy of chemotherapy in clinical practice. Several anticancer drugs exert their cytotoxic effect by triggering Fas-mediated apoptosis in some cell types. However, no investigations have been conducted to determine whether doxorubicin causes apoptosis in thyroid carcinomas. In the present study, we assessed the cytotoxic and apoptotic effects of doxorubicin on two thyroid cancer cell lines (FTC 238 and FTC 133). Cytotoxic effects of doxorubicin were evaluated by a 3-(4,5 dimethylthiazol-2yl) 2-5 diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by fluorescein isothiocyanate-conjugated annexin V/flow cytometric analysis and by DNA fragmentation. Fas expression was measured by flow cytometric analysis. After a 24-hour incubation, doxorubicin induces a dose-dependent cytotoxicity in the two cell lines. Treatment with doxorubicin (0.5 and 1 microM) for 24 hours induced cell apoptosis and upregulated Fas expression. A significant correlation was found between the fluorescence intensity values obtained with annexin V staining and those observed for Fas expression (r = 0.996; p < 0.001 or r = 0.957; 0.02 < p < 0.05 for FTC 238 or FTC 133 cells, respectively). In conclusion, doxorubicin exerts its cytotoxic effects, at least partly, through Fas-mediated apoptosis in thyroid cancer cells. These results may have clinical implications for thyroid cancer therapy.     

Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor

Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell death in vitro unless cultured in the presence of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) in which their survival is prolonged for up to 10 days. CD69 is a type II membrane antigen expressed by cytokine-activated, but not freshly isolated, PB human eosinophils. We have examined the effect of ligation of CD69 by specific monoclonal antibody (MoAb) on the viability of human eosinophils cultured with recombinant human (rh)GM-CSF. Eosinophils were purified by immunomagnetic selection and cultured with GM-CSF (10(-10) mol/L). Eighteen hours after the start of culture, a panel of CD69 MoAb or controls (anti-CR3 or isotype-matched control MoAb) were added. Viability was assessed by trypan blue exclusion and apoptosis by morphologic assessment, DNA laddering, and flow cytometric analysis of eosinophil red autofluorescence. Up to 50% of the eosinophils had undergone apoptosis 48 hours after addition of anti- CD69 MoAb compared with less than 10% apoptosis for CR3 or the isotype matched control. The majority of apoptotic eosinophils excluded trypan blue at 48 hours post CD69 ligation. More apoptotic eosinophils were observed at later time-points and this was associated with loss of viability. At 120 hours post-addition of the anti-CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable compared with 84% +/- 3.4% for the CR3 control (P < .001). A F(ab)2 fragment of CD69 MoAb P8, also induced apoptosis in GM-CSF cultured eosinophils. A more rapid induction of eosinophil apoptosis was obtained with CD69 MoAb immobilized via their Fc portions on protein-A coated plastic 96 well plates. Ligation of CD69 or CR3 resulted in the release of comparable quantities of eosinophil peroxidase at 48 hours post-ligation. These levels of EPO were consistent with the viability of these cells at 48 hours as assessed by exclusion of trypan blue. Finally, a neutralizing MoAb to TGF beta 1 had no effect on CD69-dependent apoptosis induction nor were there detectable quantities of TGF beta 1 in supernatants from GM-CSF-- cultured eosinophils ligated with CD69 or control MoAb. These results suggest that eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a result of cell signalling mediated by perturbation of CD69. This may represent an important physiologic mechanism for eosinophil removal in vivo.     

Sodium butyrate enhances Fas-mediated apoptosis of human hepatoma cells

BACKGROUND/AIMS: Human hepatoma cells have been reported to be resistant to Fas-mediated apoptosis. Sodium butyrate (SB) induced apoptosis of several cancer cells. We investigated the effects of SB on Fas-mediated apoptosis of hepatoma cells. METHODS: In hepatoma cells (HuH-6, HuH-7, Hep-G2, and PLC/PRF/5), susceptibility to Fas-mediated apoptosis and Fas expression were assessed. Caspase-3 activation and cell cycle progression were evaluated in HuH-6. A cDNA microarray assay was performed to screen the changes in the expression of mRNAs. RESULTS: Pretreatment with SB caused an enhancement of the sensitivity to anti-Fas-mediated cytotoxicity, though it did not increase the expression of Fas. The cDNA microarray assay revealed up-regulation of pro-apoptotic Bik, Bak, Bid and c-Jun N-terminal protein kinase-1, and down-regulation of anti-apoptotic Bag-1 and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitor protein. In some molecules, expression of the proteins was confirmed by Western blotting. An increase in truncated-Bid accompanying the reduction in Bid was also observed. CONCLUSIONS: SB enhances the susceptibility of hepatoma cells to anti-Fas-mediated cytotoxicity by altering the mRNA and protein expression and/or the activation status of proteins that could be involved in the Fas signaling pathway. SB may have an important role in the elimination of hepatoma cells.     

Fas-mediated apoptosis in acute alcoholic hepatitis

BACKGROUND/AIMS: The Fas-mediated apoptosis pathway has been implicated in liver diseases. The aim of the study was to investigate the role of this system in alcoholic hepatitis. METHODOLOGY: The expression of Fas, Fas ligand, and intracellular signaling molecules for apoptosis were determined by immunoblot analysis in fresh frozen liver samples from 19 patients with alcoholic liver disease. RESULTS: Fas and Fas ligand expression was significantly increased in the liver of patients with alcoholic hepatitis (n = 11) as compared with alcoholic liver disease patients without hepatitis (n = 8). Similarly, there were significant differences in the expression of FADD, ICE, and CPP32 in the liver between the two groups. There were significant positive correlations between the Fas ligand and the FADD, ICE, or CPP32 levels in the liver. The expression of Fas, Fas ligand, FADD, ICE, or CPP32 correlated with serum markers of hepatic injury. Plasma soluble Fas levels in patients with alcoholic hepatitis (median: 15.1 U/mL, range: 9.7-19.2 U/mL) were significantly higher than in normal controls (n = 9) (median: 2.8 U/mL, range: 1.9-3.7 U/mL; p < 0.001). There was a significant positive correlation between plasma soluble Fas levels and the hepatic expression of FADD in these patients. CONCLUSIONS: These results indicate that Fas-mediated apoptosis may play an important role in alcoholic hepatitis.     

Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells

BACKGROUND & AIMS: Intestinal epithelial cell apoptosis occurs continually without apparent permeability defects and is increased in response to intestinal inflammation. We hypothesized that increased, immune-mediated apoptosis during inflammation might result in barrier dysfunction of the epithelium. METHODS: T84 cells were cultured as a polarized monolayer and exposed to agonist antibody to Fas. Barrier function was assessed by transepithelial resistance and permeability measurements. Immunofluorescent staining was used to examine junctional protein expression. RESULTS: Fas expression is predominantly basolateral in polarized T84 monolayers. Basolateral cross-linking of the Fas receptor resulted in T84 cell apoptosis and a loss of 50% of the cells within 24 hours. Apoptosis was coincident with a decrease in transepithelial electrical resistance and increased flux of small but not large molecules. Preservation of barrier function was associated with dramatic rearrangement of tight junctions and desmosomal junctions in apoptotic monolayers. E-cadherin-mediated cell contact was maintained between intact cells in the monolayer, thus sealing gaps created by apoptotic cells. Apoptosis and barrier dysfunction could be prevented by caspase inhibition. CONCLUSIONS: Immune-mediated apoptosis of intestinal epithelial cells may contribute to the permeability defects associated with inflammatory conditions of the bowel, but the intestinal epithelium is remarkably resilient in the face of apoptosis.     

Fas-mediated apoptosis in accelerated graft arteriosclerosis

Pathological conditions have been recognized where vessel destruction is a prominent feature of the pathogenic process. One such condition consists of the chronic rejection of blood vessels in transplanted solid organs. Accelerated graft arteriosclerosis (AGA) is a multifactorial process characterized by the concentric proliferation of smooth muscle cells (SMCs) within the intima of the vessel wall of transplanted organs. Proliferation of SMCs within the intima corresponds to a response of these cells to injury. In situations like restenosis post-angioplasty, the mechanism of injury: the mechanical disruption of the tunica media, is evident. However, in the case of AGA, the mechanism of injury has remained elusive. In this report, we provide evidence that injury to SMCs in AGA vessels requires an intact Fas pathway. The resulting damage to the tunica media and internal elastic lamina, in turn, might trigger the proliferation of intimal smooth muscle cells that appear to be less sensitive to Fas mediated killing, particularly when supported by a favorable context of inflammatory cytokines and growth factors, as it is the case in AGA. This pathogenic process results in a absolute loss of functional blood vessels that is not being compensated by an efficient angiogenic response. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils.

We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5-mediated survival. In the absence of human IL-5 (hIL-5) mature eosinophils succumbed after 4 days, while in the presence of hIL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hIL-5, while no significant DNA fragmentation was observed in the culture with hIL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hIL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hIL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.     

Protein-tyrosine phosphorylation regulates apoptosis in human eosinophils and neutrophils.

Early signaling events that control the process of programmed cell death are largely unknown. Tyrosine phosphorylation plays a major role in transmembrane signal transduction through most cell surface receptors. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine released by activated T cells, has been shown to increase tyrosine phosphorylation in several cells and to inhibit granulocyte cell death in vitro. In this study, we demonstrate that the effect of GM-CSF on granulocyte cell death can be blocked by the tyrosine kinase inhibitor genistein, suggesting that increases in tyrosine phosphorylation are essential to inhibit cell death. To analyze the role of tyrosine phosphorylation for the regulation of granulocyte cell death more precisely, we increased levels of tyrosine phosphorylation using the protein-tyrosine phosphatase inhibitor phenylarsine oxide (PAO). Similar to GM-CSF, treatment of the cells with PAO was followed by high increases in tyrosine phosphorylation and inhibition of programmed cell death in human eosinophils and neutrophils. Strikingly, at low concentrations of the inhibitor and low induction of tyrosine phosphorylation, acceleration of apoptosis was observed. Genistein and herbimycin A reversed the effects of PAO on tyrosine phosphorylation and granulocyte apoptosis. These results suggest that programmed eosinophil and neutrophil death is regulated by early events of signal transduction pathways such as tyrosine phosphorylation.     

K1 protein of human herpesvirus 8 suppresses lymphoma cell Fas-mediated apoptosis

Expression of the K1 gene of human herpesvirus 8 activates nuclear factor-kappaB and induces lymph node hyperplasia and lymphomas in transgenic mice. To further delineate its role in cell survival, we determined whether K1 altered apoptosis of lymphoma cells. K1 protein is expressed in Kaposi sarcoma and primary effusion lymphoma. We retrovirally transfected BJAB lymphoma, THP-1, U937, and Kaposi sarcoma SLK cells to express K1 and a K1 mutant with the deleted immunoreceptor tyrosine-based activation motif (K1m). We challenged cells with an agonistic anti-Fas antibody, Fas ligand, irradiation, and tumor necrosis factor-related apoptosis-inducing ligand. K1 transfectants but not K1m transfectants exhibited reduced levels of apoptosis induced by the anti-Fas antibody but not apoptosis induced by the tumor necrosis factor-related apoptosis-inducing ligand or irradiation. K1 expression resulted in reduced apoptosis rates as shown in several assays. K1 induced a modest reduction in levels of Fas-associated death domain protein, and procaspase 8 recruited to the death-inducing signaling complex. Finally, K1 transfectants cleaved procaspase 8 at significantly lower rates than did K1m transfectants. K1-transfected mice, compared with vector-transfected mice, showed lower death rates after challenge with anti-Fas antibody. K1 may contribute to lymphoma development by stimulating cell survival by selectively blocking Fas-mediated apoptosis.     

Levocetirizine and cytokine production and apoptosis of human eosinophils.

Antihistamines are a common therapy for allergic symptoms. Eosinophilic infiltration is considered a hallmark of allergic inflammation. Eosinophils are capable of mediating airway mucosal damage by producing various inflammatory mediators including cytokines, chemokines, basic granule proteins, lipid mediators, and growth factors. Reduced eosinophil apoptosis is thought to be an important feature in the formation of eosinophilia in allergic diseases such as allergic rhinitis, atopic eczema, and asthma. The aim of this study was to investigate the effects of levocetirizine on the production of inflammatory mediators by eosinophils and on eosinophil apoptosis. The production of cytokines and other inflammatory mediators by human eosinophils was measured by a cytokine antibody array. Apoptosis of isolated human eosinophils was assessed by measuring the relative DNA content of propidium iodide-stained cells. Of the 40 cytokines studied, levocetirizine (1 microM) was found to enhance the release of tissue inhibitor of metalloproteinases 1 and 4, matrix metalloproteinase 9, and heparin-binding epidermal growth factor and to attenuate the production of interleukins (IL)-1 beta and IL-7 and stem cell factor in lipopolysaccharide-stimulated human eosinophils. Levocetirizine did not alter constitutive eosinophil apoptosis or eosinophil survival induced by IL-5, granulocyte/macrophage colony-stimulating factor, tumor necrosis factor alpha, or salbutamol. The results of this study suggest that levocetirizine modulates the profile of inflammatory mediators including cytokines, growth factors, proteinases, and antiproteinases produced by eosinophils, which may be of importance in allergic inflammation and airway remodeling. However, eosinophil longevity seems not to be modulated by levocetirizine.     

Induction of apoptosis in human eosinophils by anti-Fas antibody treatment in vitro

Fas antigen (CD95) can induce apoptosis of cells such as lymphocytes and neutrophils. To determine whether Fas antigen is involved in eosinophil apoptosis, we examined its expression and function on eosinophils in vitro. Purified human eosinophils expressed low but consistently detectable levels of Fas antigen. Culture of eosinophils in up to 10 ng/mL interleukin-5 (IL-5) prolonged eosinophil survival; incorporation of 1 to 1,000 ng/mL Fas antibody led to significant reductions in IL-5-induced eosinophil viability after 48 to 72 hours of culture. Reductions in survival could not be overcome by IL-5 and also occurred in the absence of exogenous IL-5. Preactivation of eosinophils with platelet-activating factor (PAF) significantly reduced eosinophil viability without altering the survival-reducing effects of Fas antibody treatment. In contrast, RANTES did not affect eosinophil viability or Fas antibody-induced reductions in eosinophil survival. After treatment with Fas antibody, electron microscopy of eosinophils and gel electrophoresis of DNA extracted from eosinophils demonstrated changes consistent with apoptosis. These data demonstrate that Fas antigen can modify eosinophil survival by inducing apoptosis through a pathway that is, at least in part, independent of the survival- promoting effects of IL-5.     

Sulfonylurea induced beta-cell apoptosis in cultured human islets

Loss of beta-cell mass and function raises a concern regarding the application of sulfonylureas for the treatment of type 2 diabetes because previous studies have shown that agents that cause closure of inwardly rectifying K(+) sulfonylurea receptor subtype of ATP-sensitive potassium channels, such as tolbutamide and glibenclamide, induce apoptosis in beta-cell lines and rodent islets. Therefore, we investigated the effect of the new insulin secretagogues, repaglinide and nateglinide, and the sulfonylurea, glibenclamide, on beta-cell apoptosis in human islets. Human islets from six organ donors were cultured onto extracellular matrix-coated plates and exposed to glibenclamide, repaglinide, or nateglinide. The doses of the three compounds were chosen according to detected maximal effects, i.e. efficacy. Exposure of human islets for 4 h to 0.1 and 10 microm glibenclamide induced a 2.09- and 2.46-fold increase in beta-cell apoptosis, respectively, whereas repaglinide (0.01 and 1 microm) did not change the number of apoptotic beta-cells. At low concentration (10 microm), nateglinide did not induce beta-cell apoptosis. However, at high concentration of 1000 microm, it induced a 1.49-fold increase in the number of apoptotic beta-cells. Prolonged exposure for 4 d of the islets to the secretagogues induced beta-cell apoptosis. The increase was of 3.71- and 4.4-fold at 0.1 and 10 microm glibenclamide, 2.37- and 3.8-fold at 0.01 and 1 microm repaglinide, and of 3.2- and 4.6-fold at 10 and 1000 microm nateglinide, respectively. Glibenclamide at 0.1-10 nm (doses that were less efficient on insulin secretion) did not induce beta-cell apoptosis after 4 h incubation as well as 0.1 nm after 4 d incubation. However, 1 and 10 nm glibenclamide for 4 d induced a 2.24- and 2.53-fold increase in beta-cell apoptosis, respectively. Taken together, closure of the inwardly rectifying K(+) sulfonylurea receptor subtype of ATP-sensitive potassium channels induces beta-cell apoptosis in human islets and may precipitate the decrease in beta-cell mass observed in patients with type 2 diabetes.     

COX-2 inhibits Fas-mediated apoptosis in cholangiocarcinoma cells

Fas expression has been shown to negatively regulate the progression of cholangiocarcinoma cells in xenografts. However, many human cholangiocarcinomas express Fas, suggesting these cancers have developed mechanisms to inhibit Fas-mediated apoptosis. Cyclooxygenase-2 (COX-2), which generates prostanoids, is expressed by many cholangiocarcinomas. Therefore, our aim was to determine whether COX-2 expression inhibits death receptor--mediated apoptosis in KMBC cells, a cholangiocarcinoma cell line. These cells express messenger RNA for the death receptors Fas, tumor necrosis factor receptor 1 (TNF-R1), death receptor 4 (DR4), and DR5. Agonists for these death receptors, CH-11, TNF-alpha, and TRAIL all induced apoptosis. However, COX-2, whether induced by proinflammatory cytokines or transient transfection, only significantly inhibited Fas-mediated apoptosis. The COX-2 inhibitor NS-398 restored Fas-mediated apoptosis in COX-2 transfected cells. Prostaglandin E2 reduced apoptosis and mitochondrial depolarization after treatment with the Fas agonist CH-11. Of a variety of antiapoptotic proteins examined, COX-2/prostaglandin E2 only increased expression of Mcl-1, an antiapoptotic member of the Bcl-2 family. In conclusion, these data suggest that prostanoid generation by COX-2 specifically inhibits Fas-mediated apoptosis, likely by up-regulating Mcl-1 expression. Pharmacologic inhibition of COX-2 may be useful in augmenting Fas-mediated apoptosis of cholangiocarcinoma cells.     

The role of Fas-mediated apoptosis in thyroid disease

Recent evidence has emphasized the importance of apoptosis in the maintenance of tissue homeostasis and the pathogenesis of malignant and immune diseases. Autoimmune thyroid diseases such as Hashimoto's thyroiditis and Graves' disease, as well as other autoimmune endocrine diseases, have been associated with dysregulation of apoptotic signaling pathways. In particular, dysfunction of the Fas apoptotic pathway or production of soluble factors including soluble Fas and soluble Fas ligand may be involved in the pathogenesis of these disorders. On the other hand, malignant thyroid cells may avoid Fas-mediated suicide possibly by expression of inhibitors of apoptosis and evade the immune system by inducing apoptosis on infiltrating lymphocytes. The delicate balance between cell proliferation and cell death through the Fas pathway may also play an important role in the control of thyroid cell mass and goitrogenesis. This review analyzes the current evidence on the role of Fas-mediated apoptosis in the pathogenesis of thyroid diseases including Hashimoto's thyroiditis, Graves' disease, thyroid cancer and goiter. However, the exact mechanisms involved in the regulation of apoptosis in thyroid disease remain unclear. Further investigation is needed.     

Sensitivity to Fas-mediated apoptosis is determined below receptor level in human vascular smooth muscle cells

Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1+/-2.3% viable, n = 4, P = NS) and Fas-sensitive (31.3+/-2.6% viable, n = 3, P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.     

FLIP switches Fas-mediated glucose signaling in human pancreatic beta cells from apoptosis to cell replication

Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic β cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of β cell turnover. In human islets, elevated glucose concentrations impair β cell proliferation and induce β cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic β cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive β cells; FLIP was no longer detectable in such TUNEL-positive β cells. Up-regulation of FLIP, by incubation with transforming growth factor β or by transfection with an expression vector coding for FLIP, protected β cells from glucose-induced apoptosis, restored β cell proliferation, and improved β cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human β cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.     

紫杉醇诱导人体外培养滑膜细胞凋亡

目的研究紫杉醇对类风湿关节炎（ＲＡ）的作用。方法用光镜、电镜、共聚焦显微镜、流式细胞仪及ＤＮＡ凝胶电泳等方法检测了紫杉醇诱导体外培养的９例ＲＡ与５例骨关节炎（ＯＡ）及４例正常对照（骨折）患者关节滑膜细胞凋亡（Ａｐｏ）情况。结果紫杉醇对３种体外培养的滑膜细胞均可诱导出现凋亡，其中ＲＡ组最高，可达６７０４％±１１２０％。ＯＡ组为３８２８％±４４０％，正常对照组为１５１６％±８７５％。ＲＡ组凋亡细胞百分率显著高于ＯＡ组（Ｐ＜０．０１）和正常对照组（Ｐ＜０．０１），在一定剂量范围内具有时间和浓度依赖性。结论紫杉醇可能具有临床治疗ＲＡ的潜在性价值，而诱导ＲＡ滑膜细胞凋亡可能为寻找新的ＲＡ治疗药物提供一种新途径     

碘对人甲状腺细胞凋亡的影响

目的：研究碘对人甲状腺细胞亡的影响,探讨其在甲状腺疾病发病机制中的作用和意义。方法：分别以不同浓度Nal刺激单层培养的人正常甲状腺上皮细胞,采用流式细胞术,Western印迹和半定量RT－PCR等方法分别检测刺激前后细胞凋亡率,凋亡相关蛋白Fas,Bcl-2和Bak以及Fas,可溶性Fas(sFas)mRNA表达的变化。并以ELISA法检测细胞培养液中sFas的含量。结果：（1）低浓度NaI(10^-8mol/L)明显抑制细胞凋亡（P＜0．05）,中,高浓度（10^-6-10^-4mol/L)时显著增加细胞凋亡（P＜0．05）;（2）低浓度NaI显著促进sFas蛋白及mRNA的表达（P＜．05）,不影响Fas蛋白及mRNA的表达;（3）中、高浓度NaI则明显增加Fas蛋白及mRNA表达,但抑制sFas蛋白及mRNA表达（P＜0．05）;（4）在10^-8-10^-4mol/L浓度范围内,NaI剂量依赖性地抑制甲状腺上皮细胞表达Bcl-2蛋白,但显著增加Bak蛋白的表达（P＜0．05）,结论：碘可通过调节Fas,sFas,Bcl-2及Bak的表达,影响甲状腺细胞凋亡的发生,其中低浓度碘可抑制凋亡,而中,高浓度碘则促进细胞凋亡。