Interleukin-1beta and tumor necrosis factor (TNF)-alpha sensitize human thyroid epithelial cells to TNF-related apoptosis-inducing ligand-induced apoptosis through increases in procaspase-7 and bid, and the down-regulation of p44/42 mitogen-activated protein kinase activity

Primary thyroid cells are resistant to TNF-related apoptosis-inducing ligand (TRAIL). Previously we showed that the combination of IL-1beta and TNFalpha facilitated TRAIL-mediated apoptosis in these cells and enhanced cell surface expression of TRAIL receptors. The aim of this study was to further characterize the mechanism by which these cytokines sensitized primary thyroid cells to TRAIL-mediated apoptosis. IL-1beta and TNFalpha increased the concentrations of procaspase-7 and Bid. In contrast, the p44/42 MAPK (Erk) pathway was active in thyroid cells and this activity was significantly decreased after exposure to IL-1beta/TNFalpha. A MAPK kinase inhibitor (U0126) could enhance the cytokine-induced sensitization of thyroid cells to TRAIL, reinforcing the inhibitory role of Erk on TRAIL signaling. In conclusion, IL-1beta/TNFalpha treatment sensitizes human thyroid cells to TRAIL-mediated apoptosis through increased surface expression of TRAIL receptors, increased expression of procaspase-7 and Bid, and the inhibition of p44/42 MAPK (Erk) pathway.     

TNF-related apoptosis-inducing ligand death pathway-mediated human beta-cell destruction

AIMS/HYPOTHESIS: The aim of this study is to investigate whether apoptosis in human beta cells can be related to the induction of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. METHODS: We examined the expression of TRAIL and TRAIL receptors in two human pancreatic beta-cell lines and in human primary islet cells using RT-PCR assays and flow cytometric analyses and tested TRAIL-mediated beta-cell destruction in (51)Cr release cytotoxicity assays, Annexin-V and APO-DIREC assays. RESULTS: Most of the human beta cells express TRAIL receptors-R1, -R2, -R3, -R4 and/or TRAIL. TRAIL induced much stronger cytotoxicity and apoptosis to beta-cell lines CM and HP62 than did FasL, TNF-alpha, LTalpha1beta2, LTalpha2beta1, LIGHT, and IFN-gamma. The cytotoxicity and apoptosis induced by TRAIL to beta-cell lines CM were inhibited competitively by soluble TRAIL receptors, R1, R2, R3 or R4. Treatment of these beta cells with antibodies against TRAIL receptors was able to block the cytotoxicity of TRAIL to these cells. Beta-cell antigen-specific CTL (CD4(+) and CD8(+)) clones express TRAIL, suggesting that these cells are potential sources of TRAIL-inducing beta-cell destruction. Normal primary islet cells from most donors are resistant to the cytotoxicity mediated by TRAIL. However, treatment with an inhibitor of protein synthesis (cycloheximide) or with an enzyme (PI-PLC) that can remove TRAIL-R3 from the islet-cell membrane was able to increase the susceptibility of TRAIL-resistant primary islet cells to the TRAIL death pathway. CONCLUSION/INTERPRETATION: The TRAIL death pathway is present and can function in human islet beta cells, but unidentified inhibitors of the TRAIL death pathway are present in normal islet cells.     

The role of apoptosis-inducing receptors of the tumor necrosis factor family in thyroid cancer

The tumor necrosis factor (TNF) family comprises several ligands, such as the prototype TNF-alpha, the Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), which trigger apoptosis in susceptible cells by activating respective cell-surface receptors. The study of these cell death pathways has attracted significant attention in several fields, including that of thyroid cancer, because they participate in immune system function, as an arm of cell-mediated cytotoxicity, and because recombinant ligands are available for pharmacological use. TNF-alpha is a pluripotent cytokine that induces both pro-apoptotic and anti-apoptotic effects on thyroid carcinoma cells. FasL triggers apoptosis in other tumor types, but thyroid carcinoma cells are resistant to this effect. On the other hand, TRAIL potently and selectively kills thyroid carcinoma cells. Consequently, TRAIL is the only member of the family with significant anticancer activity and an acceptable toxicity profile to be used as a novel therapy for thyroid cancer. The caspase inhibitor FLIP plays a significant role in negatively regulating receptor-induced apoptosis. Thelper 1-type cytokines, such as interferon-gamma, TNF-alpha and interleukin-1beta increase the sensitivity of both normal and neoplastic thyrocytes to FasL and TRAIL. On the other hand, IGF-I and other growth/survival factors produced in the local tumor microenvironment activate the phosphatidylinositol 3-kinase/Akt kinase pathway and exert an anti-apoptotic effect by upregulating several apoptosis inhibitors, including FLIP. Pharmacological modulation of apoptosis induced by FasL and TRAIL/Apo2L holds promise of therapeutic applications in human malignancies.     

Apoptosis induced by FasL and TRAIL/Apo2L in the pathogenesis of thyroid diseases.

FasL and TRAIL/Apo2L participate in cell-mediated cytotoxicity by inducing apoptosis in susceptible cells via respective cell surface receptors. Normal and neoplastic thyroid tissues are resistant to FasL-induced apoptosis but are sensitized by Th-1-type cytokines. In Hashimoto's thyroiditis, both FasL and its receptor, Fas, are strongly upregulated and their interaction leads to the suicidal/fratricidal death of thyrocytes. In Graves' disease, FasL expression in thyroid follicular cells is induced by thionamides and kills infiltrating lymphocytes. In this condition, Th-2-type cytokines upregulate the anti-apoptotic molecules FLIP and Bcl-x(L) and protect thyrocytes from apoptosis. FasL is expressed by neoplastic thyrocytes and induces apoptosis of infiltrating lymphocytes. TRAIL/Apo2L kills thyroid carcinoma cells but spares normal thyrocytes, thus providing a potential therapy for thyroid cancer.     

Regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by DJ-1 in thyroid cancer cells

DJ-1, a cancer-associated protein protects cells from multiple toxic stresses. The expression of DJ-1 and its influence on thyroid cancer cell death has not been investigated so far. We analyzed DJ-1 expression in human thyroid carcinoma cell lines and the effect of DJ-1 on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. DJ-1 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downregulation of its levels significantly sensitized thyroid carcinoma cells to TRAIL-induced apoptosis, whereas the forced exogenous expression of DJ-1 significantly suppressed cell death induced by TRAIL. We also report here that TRAIL-induced thyroid cancer cell apoptosis is mediated by oxidative stress and that DJ-1, a potent nutritional antioxidant, protects cancer cells from apoptosis at least in part by impeding the elevation of reactive oxygen species levels induced by TRAIL and impairing caspase-8 activation. Subsequently, we investigated DJ-1 expression in 52 normal and 74 primary thyroid carcinomas from patients of China Medical University. The protein was not detectable in the 52 specimens of normal thyroid, while 70 out of 74 analyzed carcinomas (33 out of 33 follicular, 17 out of 19 papillary, 12 out of 13 medullar, and 8 out of 9 anaplastic) were clearly positive for DJ-1 expression. Our data demonstrated that DJ-1 is specifically expressed in thyroid carcinomas and not in the normal thyroid tissue. In addition, the protein modulates the response to TRAIL-mediated apoptosis in human neoplastic thyroid cells, at least partially through its antioxidant property.     

Proteasome inhibitors synergize with tumor necrosis factor-related apoptosis-induced ligand to induce anaplastic thyroid carcinoma cell death

CONTEXT: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive types of cancer characterized by complete refractoriness to multimodal treatment approaches. Therapeutic strategies based on the simultaneous use of proteasome inhibitors and death receptor ligands have been shown to induce apoptosis in several tumor types but have not yet been explored in ATC. OBJECTIVE AND METHODS: The aim of this study was to investigate the ability of the proteasome inhibitor Bortezomib to induce apoptosis in ATC cell lines. Bortezomib was used as a single agent or in combination with TNF-related apoptosis-induced ligand (TRAIL), a member of the TNF family that selectively induces tumor cell apoptosis. The molecular effects of Bortezomib were investigated by analyzing the expression of key regulators of cell cycle and apoptosis and the activation of different apoptotic pathways. RESULTS: Bortezomib induced apoptosis in ATC cells at doses achieved in the clinical setting, differently from conventional chemotherapeutic agents. Simultaneous treatment with low doses of Bortezomib and TRAIL had a synergistic effect in inducing massive ATC cell apoptosis. Bortezomib increased the expression of cytotoxic TRAIL receptors, p21 (WAF/CIP1) and proapoptotic second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI, and reduced the expression of antiapoptotic mediators such as cellular Fas-associated death domain-like IL-1beta converting enzyme inhibitory protein, Bcl-2, Bcl-X(L), and inhibitor of apoptosis-1, thus resulting in cell death induction through the mitochondrial apoptotic pathway. CONCLUSIONS: The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma.     

Involvement of glyceraldehyde-3-phosphate dehydrogenase in tumor necrosis factor-related apoptosis-inducing ligand-mediated death of thyroid cancer cells

TNF-related apoptosis-inducing ligand (TRAIL) is cytotoxic to most thyroid cancer cell lines, including those originating from anaplastic carcinomas, implying TRAIL as a promising therapeutic agent against thyroid cancers. However, signal transduction in TRAIL-mediated apoptosis is not clearly understood. In addition to its well-known glycolytic functions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein, including its surprising role as a mediator for cell death. In this study we explored the involvement of GAPDH in TRAIL-mediated thyroid cancer cell death. In follicular undifferentiated thyroid cells, S-nitrosylation and nuclear translocation of GAPDH appear to mediate TRAIL-induced cell death at least partially, as evidenced by pretreatment with N-nitro-L-arginine methyl ester, a competitive nitric oxide synthase inhibitor that partially but significantly attenuated TRAIL-induced apoptosis through the reduction of S-nitrosylation and nuclear translocation of GAPDH. In addition, GAPDH small interfering RNA partially prevented the apoptotic effect of TRAIL, although TRAIL-induced nitric oxide synthase stimulation and production of nitric oxide were not attenuated. Furthermore, nuclear localization of GAPDH was observed in another thyroid cancer cell line, KTC2, which is also sensitive to TRAIL, but not in those TRAIL insensitive cell lines: ARO, KTC1, and KTC3. These data indicate that nitric oxide-mediated S-nitrosylation of GAPDH and subsequent nuclear translocation of GAPDH might function as a mediator of TRAIL-induced cell death in thyroid cancer cells.     

Chelerythrin activates caspase-8, downregulates FLIP long and short,and overcomes resistance to tumour necrosis factor-related apoptosis-inducing ligand in KG1a cells

KG1a cells (CD34+/38-) express FAS and TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand) receptors but are resistant to FAS-ligand and TRAIL/APO2-L (apoptosis antigen-2 ligand)-induced apoptosis. KG1a cells are sensitized to FAS-induced apoptosis by chelerythrin, an inhibitor of protein kinase C (PKC). As cytoplasmatic adaptor molecules of FAS, e.g. FLIP [Fas-associated death domain protein (FADD)-like interleukin 1 beta-converting enzyme [FLICE (caspase-8)-inhibitory protein]], also modulate TRAIL signals, we determined whether chelerythrin affected TRAIL-mediated apoptosis. Chelerythrin by itself induced apoptosis in KG1a cells, and apoptosis was associated with activation of caspase-8. While TRAIL alone failed to activate caspase-8 or induce apoptosis, the addition of TRAIL to chelerythrin-treated cells significantly enhanced cleavage of caspase-8 and apoptosis. Chelerythrin-pretreated KG1a cells showed decreased phosphorylation of protein kinase C (PKC)-zeta and downregulation of both FLIP long and FLIP short proteins. Downregulation of FLIP and induction of apoptosis were partially abrogated by pretreatment with the specific caspase-8 inhibitor, Z-IETD-FMK. The decrease in FLIP protein expression induced by chelerythrin was accompanied by a progressive increase in mRNA levels of both FLIP long and FLIP short. CD34+ precursors from normal human marrow were also sensitive to chelerythrin but, in contrast to KG1a cells, were not sensitized to TRAIL-mediated apoptosis. Thus, resistance to TRAIL-induced apoptosis in leukaemic KG1a cells but not in normal CD34+ precursors was overcome in the presence of chelerythrin. The mechanism appeared to involve inhibition of PKC. Central targets were FLIP long and FLIP short, and their interactions with caspase-8. Whether such a pathway can be exploited to selectively target leukaemic progenitor cells remains to be determined.     

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

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

NFkappaB-mediated upregulation of bcl-xl restrains TRAIL-mediated apoptosis in murine viral hepatitis

Inhibition of NFkappaB enhances the susceptibility of cancer to TRAIL-mediated apoptosis and is suggested as a strategy for cancer therapy. Because the role of NFkappaB in TRAIL-mediated apoptosis of hepatocytes is unknown, we investigated the influence of NFkappaB-inhibition in death ligand-mediated apoptosis in hepatitis. Adenoviral hepatitis resulted in upregulation of NFkappaB-activity, which could be inhibited by expression of IkappaBalpha-superrepressor. We treated mice after the onset of adenoviral hepatitis with adenoviruses expressing FasL (AdFasL), TRAIL (AdTRAIL), or GFP (AdGFP). In contrast to apoptosis induced by AdFasL, NFkappaB inhibition strongly enhanced AdTRAIL-mediated apoptosis of hepatocytes. Expression of IkappaBalpha inhibits adenoviral infection-mediated overexpression of bcl-xl, providing a molecular mechanism for TRAIL sensitization. In agreement with this hypothesis, downregulation of bcl-xl by siRNA enhanced susceptibility of hepatocytes to TRAIL, but not to FasL-mediated apoptosis, resulting in TRAIL-mediated severe liver damage after AdTRAIL application. Our data demonstrate that inhibition of NFkappaB in adenoviral hepatitis strongly sensitizes hepatocytes to TRAIL-mediated apoptosis. Bcl-xl, in contrast to bcl-2 and c-FLIP, is strongly upregulated after viral infection and represents an essential NFkappaB-dependent survival factor against TRAIL-mediated apoptosis. In conclusion, inhibition of NFkappaB or bcl-xl during TRAIL therapy may harbor a risk of liver damage in patients with viral hepatitis.     

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.     

Apoptotic and survival signaling mediated through death receptor members during metamorphosis in the African clawed frog Xenopus laevis

The tumor necrosis factor (TNF) superfamily includes death receptor (DR) ligands, such as TNF-α, FasL, and TRAIL. Death receptors (DRs) induce intracellular signaling upon engagement of their cognate DR ligands, either leading to apoptosis, survival, or proinflammatory responses. The DR signaling is mediated by the recruitment of several death domain (DD)-containing molecules such as Fas-associated death domain (FADD) and receptor-interacting protein (RIP) 1. In this review, we describe DR signaling in mammals, and describe recent findings of DR signaling during metamorphosis in the African clawed frog Xenopus laevis. Specifically, we focus on the cell fate (apoptosis or survival) mediated through a DR ligand, TNF-α or TRAIL in endothelial cells or red blood cells (RBCs). In addition, we discuss relationships between thyroid hormone-induced metamorphosis and DR signaling.     

Modulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by chemotherapy in thyroid cancer cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many human cancer cells but not in normal cells. Thyroid cancer cells, however, appear to be relatively resistant to TRAIL-induced apoptosis. We therefore investigated the effect of chemotherapy on TRAIL-induced apoptosis in thyroid cancer cells. We used six thyroid cancer cell lines: TPC-1, FTC-133, FTC-236, FTC-238, XTC-1, and ARO82-1. We used flow cytometry to measure apoptosis, dimethyl-thiazol-diphenyltetrazolium bromide (MTT) assay to measure antiproliferation effects and Western blot to determine the expression of Bcl family proteins. Troglitazone, paclitaxel, geldanamycin, and cycloheximide were used for pretreatment. We used the Student's t test and analysis of variance (ANOVA) for statistical analysis. All thyroid cancer cell lines, except the TPC-1 cell line, were resistant to TRAIL, and growth inhibition was less than 20% at concentration of 800 ng/mL of TRAIL. In both TPC-1 (TRAIL-sensitive) and FTC-133 (TRAIL-resistant) thyroid cancer cell lines, pretreatment with troglitazone, cycloheximide, and paclitaxel enhanced TRAIL-induced cell death significantly but pretreatment with geldanamycin did not. There were no significant changes in Bcl-2, Bcl-xl, and Bax protein expression after troglitazone treatment. In conclusion, TRAIL in combination with troglitazone, paclitaxel, and cycloheximide induces apoptosis in thyroid cancer cells at suboptimal concentrations that cannot be achieved using TRAIL alone.     

Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced both cytotoxic (apoptosis) and cytostatic (cell cycle perturbation) effects on the human myeloid K562 cell line. TRAIL stimulated caspase 3 and nitric oxide synthase (NOS) activities, and both pathways cooperate in mediating inhibition of K562 survival/growth. This was demonstrated by the ability of z-VAD-fmk, a broad inhibitor of effector caspases, and N-nitro-L-arginine methyl ester (L-NAME), an NOS pharmacologic inhibitor, to completely (z-VAD-fmk) or partially (L-NAME) suppress the TRAIL-mediated inhibitory activity. Moreover, z-VAD-fmk was able to block TRAIL-mediated apoptosis and cell cycle abnormalities and increase of NOS activity. The addition of the NO donor sodium nitroprusside (SNP) to K562 cells reproduced the cytostatic effect of TRAIL without inducing apoptosis. When TRAIL was associated to SNP, a synergistic increase of apoptosis and inhibition of clonogenic activity was observed in K562 cells as well as in other myeloblastic (HEL, HL-60), lymphoblastic (Jurkat, SupT1), and multiple myeloma (RPMI 8226) cell lines. Although SNP greatly augmented TRAIL-mediated antileukemic activity also on primary leukemic blasts, normal erythroid and granulocytic cells were less sensitive to the cytotoxicity mediated by TRAIL with or without SNP. These data indicate that TRAIL promotes cytotoxicity in leukemic cells by activating effector caspases, which directly lead to apoptosis and stimulate NO production, which mediates cell cycle abnormalities. Both mechanisms seem to be essential for TRAIL-mediated cytotoxicity.     

Transforming growth factor beta can mediate apoptosis via the expression of TRAIL in human hepatoma cells

Transforming growth factor beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. However, the exact mechanism through which TGF-beta induces cell death is still unknown. We examined a potential role of various death receptor/ligand systems in TGF-beta-induced apoptosis and identified the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a mediator of TGF-beta-induced apoptosis in hepatoma cells. TGF-beta-induced apoptosis is significantly impaired upon blockage of TRAIL. We show that TRAIL is upregulated in hepatoma cells upon treatment with TGF-beta, whereas TRAIL receptor levels remain unchanged. In conclusion, our results provide evidence that the TRAIL system is critically involved in TGF-beta-induced cell death in liver pathology.     

Hepatitis B virus enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity by increasing TRAIL-R1/death receptor 4 expression

BACKGROUND/AIMS: Apoptosis by death receptors, such as Fas and tumor necrosis factor (TNF)-alpha receptor-1, play a significant role in the pathogenesis of hepatitis B virus (HBV)-infections. Although liver also expresses death receptors for TNF-related apoptosis-inducing ligand (TRAIL), information is lacking regarding the effects of HBV on apoptosis by TRAIL. Thus, the aims of this study were to examine the effects of HBV replication on TRAIL cytotoxicity. METHODS: Hep G2 and Hep G2.215 cells, the latter which is stably transfected with HBV, were employed for these studies. RESULTS: TRAIL-mediated cell killing was concentration-dependent and greater in Hep G2.2.15 cells at all doses as compared to the parent cell line, Hep G2 cells. Cell death by apoptosis was confirmed by demonstrating caspase activation and inhibition of cell killing by a caspase inhibitor, zVAD-fmk. TRAIL-R1/DR4 protein expression was enhanced in Hep G2.2.15 cells as compared to Hep G2 cells. Lamivudine treatment reduced TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression in Hep G2.2.15 cells. In Hep G2 cells transfected with the HBV-encoded X antigen (HBxAg), sensitivity to TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression were both increased. CONCLUSIONS: TRAIL-induced apoptosis is enhanced by the level of HBV replication in human hepatocytes, in part, by HBxAg-dependent upregulation of TRAIL-R1/DR4.     

Chemical sensitization and regulation of TRAIL-induced apoptosis in a panel of B-lymphocytic leukaemia cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) effectively kills tumour cells but not normal cells. We investigated TRAIL sensitivity and the TRAIL-induced apoptosis signalling pathway in a panel of B-lymphocytic leukaemia cell lines. Depending upon TRAIL sensitivity, leukaemia cells could be divided into three groups: highly sensitive, moderately sensitive and resistant. TRAIL receptor-2 (DR5) plays an important role in transducing apoptosis signals. DR5 was internalized into the cytoplasm where it recruited FAS-associated death domain protein (FADD) under TRAIL stimulation in both sensitive and resistant cells. However, the active form of caspase-8 was recruited to FADD and only sensitive cells showed increased caspase-8 activity upon TRAIL stimulation. The caspase-8 specific inhibitor, Z-IETD, impaired caspase-8 activation and completely abrogated TRAIL-induced apoptosis. These results suggest that TRAIL resistance in B-lymphocytic leukaemia cells is due to negative regulation at the level of caspase-8 activation and that caspase-8 activation is an indispensable process in TRAIL-induced apoptosis. However, FADD-like interleukin-1 beta-converting enzyme inhibitory protein (c-FLIPL) was similarly expressed and down-regulated after TRAIL stimulation in both sensitive and resistant cells. Interestingly, in some cell lines, TRAIL sensitivity and caspase-8 activity was enhanced or restored with the treatment of cycloheximide (CHX). In addition, X-linked inhibitor of apoptosis (XIAP) levels decreased significantly and rapidly following treatment with CHX. Down-regulation of XIAP may be responsible for enhancement or restoration of TRAIL sensitivity after CHX treatment in B-lymphocytic leukaemia cells.     

Human herpesvirus 7 induces the functional up-regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) coupled to TRAIL-R1 down-modulation in CD4(+) T cells

Human herpesvirus 7 (HHV-7) is endemic in the adult human population. Although HHV-7 preferentially infects activated CD4(+) T lymphocytes, the consequence of T-cell infection for viral pathogenesis and immunity are still largely unknown. HHV-7 infection induces apoptosis mostly in uninfected bystander cells but not in productively infected CD4(+) T cells. To dissect the underlying molecular events, the role of death-inducing ligands belonging to the tumor necrosis factor (TNF) cytokine superfamily was investigated. HHV-7 selectively up-regulated the expression of TNF-related apoptosis-inducing ligand (TRAIL), but not that of CD95 ligand or TNF-alpha in lymphoblastoid (SupT1) or primary activated CD4(+) T cells. Moreover, in a cell-to-cell-contact assay, HHV-7-infected CD4(+) T lymphocytes were cytotoxic for bystander uninfected CD4(+) T cells through the TRAIL pathway. By contrast, HHV-7 infection caused a marked decrease of surface TRAIL-R1, but not of TRAIL-R2, CD95, TNF-R1, or TNF-R2. Of note, the down-regulation of TRAIL-R1 selectively occurred in cells coexpressing HHV-7 antigens that became resistant to TRAIL-mediated cytotoxicity. These findings suggest that the TRAIL-mediated induction of T-cell death may represent an important immune evasion mechanism of HHV-7, helping the virus to persist in the host organism throughout its lifetime.     

Expression of TNF-related apoptosis inducing ligand (TRAIL) on infiltrating cells and of TRAIL receptors on salivary glands in patients with Sjögren's syndrome

BACKGROUND: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis of tumor cells but not normal cells; its role in normal non-transformed tissues is unknown. OBJECTIVE: To evaluate the role of apoptosis mediated by TRAIL and TRAIL-receptor (TRAIL-R) system in lymphocytic sialadenitis in patients with Sj?gren's syndrome. METHODS: The expression of TRAIL and TRAIL-R1, 2, 3 and 4 in lymphocytic sialadenitis was examined by immunoperoxidase staining in patients with Sj?gren's syndrome and in normal subjects. To elucidate the mechanism of de novo expression of TRAIL-R1 antigen, we quantitatively investigated its induction by cytokines in human salivary duct cell line (HSG) by cell enzyme-linked immunosorbent assay. In human salivary duct cells stimulated by cytokines, we investigated the induction of apoptotic cell death by recombinant TRAIL protein. RESULTS: In patients with massive mononuclear cell infiltration, some infiltrating cells showed TRAIL. In patients with severe lymphocytic sialadenitis, TRAIL-R1, TRAIL-R2, or both were strongly expressed on the ductal epithelial cells. Neither TRAIL-R3 nor R4 were observed on ductal epithelium. In contrast, TRAIL-R1 and R2 were not found in the minor salivary glands of normal subjects or patients with mild lymphocytic sialadenitis. Unstimulated HSG cells did not express TRAIL-R1. Interferon-gamma (IFN-gamma) consistently upregulated levels of TRAIL-R1. In contrast, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1 beta), IL-2, and IL-4 had no effect on TRAIL-R1 levels. HSG cells expressing TRAIL-R1 in response to IFN-gamma were susceptible to apoptosis by recombinant TRAIL protein. CONCLUSION: Our findings suggest that TRAIL and TRAIL-R system may play a role in the pathogenesis of lymphocytic sialadenitis in patients with Sj?gren's syndrome.     

Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells

AIM: To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylori) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in H pylori-infected gastric mucosa. METHODS: Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture. Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death. Infiltrating lymphocytes were isolated from H pylori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry. RESULTS: The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pylori alone. Interestingly, the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H pylori (Control vs TRAIL and H pylori: 0.51+/-0.06 vs 2.29+/-0.27, P = 0.018). A soluble TRAIL receptor (DR4-Fc) could specifically block the TRAIL-mediated apoptosis. Further studies demonstrated that infiltrating T-cells in gastric mucosa expressed TRAIL on their surfaces, and the induction of TRAIL sensitivity by H pylori was dependent upon direct cell contact of viable bacteria, but not CagA and VacA of H pylori. CONCLUSION: H pylori can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis. Modulation of host cell sensitivity to apoptosis by bacterial interaction adds a new dimension to the immunopathogenesis of H pylori infection.