Fas-associated Death Domain-like Interleukin-1beta-converting Enzyme Research Articles

Deletion of Kaposi's Sarcoma-Associated Herpesvirus FLICE Inhibitory Protein, vFLIP, from the Viral Genome Compromises the Activation of STAT1-Responsive Cellular Genes and Spindle Cell Formation in Endothelial Cells

Kaposi's sarcoma herpesvirus (KSHV) Fas-associated death domain (FADD)-like interleukin-1 beta-converting enzyme (FLICE)-inhibitory protein, vFLIP, has antiapoptotic properties, is a potent activator of the NF-κB pathway, and induces the formation of endothelial spindle cells, the hallmark of Kaposi's sarcoma, when overexpressed in primary endothelial cells. We used a reverse genetics approach to study several functions of KSHV vFLIP in the context of the whole viral genome. Deletion of the gene encoding vFLIP from a KSHV genome cloned in a bacterial artificial chromosome (BAC) reduced the ability of the virus to persist and induce spindle cell formation in primary human umbilical vein endothelial cells (HUVECs). Only a few, mainly interferon (IFN)-responsive, genes were expressed in wild-type KSHV (KSHV-wt)-infected endothelial cells at levels higher than those in KSHV-ΔFLIP-infected endothelial cells, in contrast to the plethora of cellular genes induced by overexpressed vFLIP. In keeping with this observation, vFLIP induces the phosphorylation of STAT1 and STAT2 in an NF-κB-dependent manner in endothelial cells. vFLIP-dependent phosphorylation of STAT1 and STAT2 could be demonstrated after endothelial cells were infected with KSHV-wt, KSHV-ΔFLIP, and a KSHV-vFLIP revertant virus. These findings document the impact of KSHV vFLIP on the transcriptome of primary endothelial cells during viral persistence and highlight the role of vFLIP in the activation of STAT1/STAT2 and STAT-responsive cellular genes by KSHV.

Akt promotes chemoresistance in human ovarian cancer cells by modulating cisplatin-induced, p53-dependent ubiquitination of FLICE-like inhibitory protein

Although Akt is a determinant of cisplatin (cis-diaminedichloroplatinum (CDDP)) resistance in ovarian cancer cells, which is related in part to its inhibitory action on p53 activation, precisely how Akt confers CDDP resistance is unclear. In this study, we show that CDDP induced p53-dependent Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP) degradation in chemosensitive ovarian cancer cells but not their resistant counterparts. CDDP induced FLIP-p53-Itch interaction, colocalization and FLIP ubiquitination in chemosensitive but not chemoresistant ovarian cancer cells. Moreover, although activated Akt inhibited CDDP-induced FLIP degradation and apoptosis in sensitive cells, these responses were facilitated by dominant-negative Akt expression in chemoresistant cells. Inhibition of Akt function also facilitated p53-FLIP interaction and FLIP ubiquitination, which were attenuated by p53 silencing. These results suggest that Akt confers resistance, in part, by modulating CDDP-induced, p53-dependent FLIP ubiquitination. Understanding the precise etiology of chemoresistance may improve treatment for ovarian cancer.

FOXO3a mediates the androgen-dependent regulation of FLIP and contributes to TRAIL-induced apoptosis of LNCaP cells

Androgen-withdrawal-induced apoptosis (AWIA) is deregulated in androgen refractory prostate cancer. Androgens have been shown to positively regulate expression of the antiapoptotic FADD-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP), and reduced FLIP expression precedes apoptosis after androgen withdrawal. Here, we show that FLIP protein expression is downregulated in castrated rats, while in LNCaP cells, androgens regulate FLIP in a manner that is dependent on phosphoinositol-3-kinase (PI3K) and Akt signaling. Specifically, treatment of LNCaP cells with LY294002, or expression of either PTEN or a non-phosphorylatable form of FOXO3a (FOXO3aTM), downregulates FLIP protein and mRNA. Conversely, treatment with androgens in the absence of PI3/Akt signaling, or following expression of FOXO3aTM, leads to increased FLIP expression. A FOXO3a binding site was identified in the FLIP promoter and shown necessary for the combined effects of androgens and FOXO3a on FLIP transcription. FOXO3a binds the androgen receptor, suggesting that the transcriptional synergy depends on an interaction between these proteins. Finally, LNCaP cells are sensitized to TRAIL-induced apoptosis by PTEN or LY294002, and rescued by androgens. FOXO3aTM also sensitizes cells to androgen-inhibited TRAIL apoptosis. Androgen rescue was diminished when either FOXO3a or FLIP was reduced by siRNA. These data support a role for FOXO3a in AWIA.

Expression of c-FLIP in Gastric Cancer and its Relation to Tumor Cell Proliferation and Apoptosis

BackgroundThe expression of c-FLIP (cellular Fas-associated death domain-like interleukin-1 β-converting enzyme (FLICE)-inhibitory protein), which is a member of the family of inhibitors of apoptosis, has been associated with tumor development and progression. The aim of this study was to evaluate the expression of c-FLIP in gastric cancer and its correlation with tumor cell proliferation, apoptosis and the clinicopathologic features.MethodsImmunohistochemical staining with anti-c-FLIP antibody was performed in 98 tissue samples obtained from gastric cancer patients who underwent surgical treatment. The apoptotic cells were visualized by terminal deoxynucleotidyl transferase (TdT) mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL), and the proliferative cells were visualized by staining with Ki-67 antibody.ResultsThe positive expression of c-FLIP in the gastric cancer tissues was demonstrated in 57.1% of the cases. The expression of c-FLIP was increased in the gastric cancer tissues compared with the matched normal gastric mucosa. The expression of c-FLIP was significantly associated with histologic differentiation (p=0.038). However, there was no association between the c-FLIP expression and the other clinicopathological parameters, including patient survival. The Ki-67 labeling index (KI) for the 98 tumors ranged from 7.6 to 85.0 with a mean KI of 50.4±15.7. The mean KI value of the c-FLIP positive tumors was 54.1±15.3 and this was significantly higher than that of the c-FLIP negative tumors (p=0.005). The apoptotic index (AI) for the 98 tumors ranged from 0.0 to 10.0 with a mean AI of 7.4±2.3. There was no significant difference between the c-FLIP expression and the AI (p=0.347).ConclusionsThese results suggest that the c-FLIP expression may be associated with tumor cell proliferation of gastric cancer.

CFLIP expression correlates with tumour progression and patient outcome in non‐Hodgkin lymphomas of low grade of malignancy

The present study investigated whether the expression of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (cFLIP) conveys prognostic information in non-Hodgkin lymphomas (NHLs). cFLIP expression was quantified by immunohistochemistry and immunofluorescence in biopsy specimens from 86 NHL patients for whom clinical information was available. NHL malignancy was graded as high/intermediate or low according to the World Health Organization Classification of Lymphoid Neoplasms. cFLIP was positive in 23 of 45 high-/intermediate-grade NHLs and in 25 of 41 low-grade NHLs. Negative expression of cFLIP was associated with the presence of apoptotic cells in the tumour mass, regardless of the histotype and of the malignancy grade. In NHLs positive for cFLIP, 11 of 23 (48%) high-/intermediate-grade cases and 18 of 25 (72%) low-grade cases showed a bad outcome. In NHLs negative for cFLIP, only four of 22 (18%) high-/intermediate-grade patients and 12 of 16 (75%) low-grade patients achieved complete remission. All these correlations were statistically significant. The correlation of cFLIP expression with clinical outcome was independent of therapy, whether or not it included anti-CD20 antibody (Rituximab). The present findings strongly indicate that cFLIP is a reliable predictor of tumour progression and clinical prognosis in NHLs of low grade of malignancy.

The KSHV oncoprotein vFLIP contains a TRAF‐interacting motif and requires TRAF2 and TRAF3 for signalling

Primary effusion lymphomas (PELs) characterized by infection with the Kaposi's sarcoma herpesvirus (KSHV; also called human herpesvirus 8) depend on the expression of the viral FADD-like interleukin-1-beta-converting enzyme (FLICE)/caspase-8-inhibitory protein (vFLIP) for their survival. This effect is achieved by activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Tumour necrosis factor (TNF) receptor-associated factors (TRAFs) are direct mediators of NF-kappaB signalling by TNF family receptors and the Epstein-Barr virus oncoprotein latent membrane protein 1 and so we assessed the role of TRAFs in signalling by vFLIP. Here, we report the identification of a TRAF-interacting motif (PYQLT) in vFLIP, which is not present in other FLIP molecules. We show that vFLIP directly binds to TRAF2 in vitro and in PEL cells. TRAF2 and TRAF3 are required for induction of NF-kappaB and associated cell survival, as well as Jun amino-terminal kinase phosphorylation by vFLIP, whereas TRAF1, TRAF5 and TRAF6 are dispensable. Mutations in the P93 or Q95 amino acids within the TRAF-interacting motif of vFLIP abolish its ability to bind to TRAF2 and to signal to NF-kappaB. TRAF2, but not TRAF3, mediates the association of vFLIP with the IkappaB kinase complex. These data indicate that vFLIP uses TRAF2 and TRAF3 for signalling to NF-kappaB, which is crucial for KSHV-associated lymphomagenesis.

Expression of Cellular FLICE Inhibitory Proteins (cFLIP) in Normal and Traumatic Murine and Human Cerebral Cortex

Cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins (cFLIPs) are endogenous caspase homologues that inhibit programmed cell death. We hypothesized that cFLIPs are differentially expressed in response to traumatic brain injury (TBI). cFLIP-alpha and cFLIP-delta mRNA were expressed in normal mouse brain-specifically cFLIP-delta (but not cFLIP-alpha) protein was robustly expressed. After controlled cortical impact (CCI), cFLIP-alpha expression increased initially then decreased to control levels at 12 h, increasing again at 24-72 h (P<0.05). cFLIP-delta expression was decreased in brain homogenates by 12 h after CCI, then increased again at 24 to 72 h (P<0.05). cFLIP-delta immunostaining was markedly reduced in injured cortex, but not hippocampus, at 3 to 72 h after CCI. In cortex, reduced cFLIP-delta staining was found in TUNEL-positive cells, but in hippocampus TUNEL-positive cells expressed cFLIP-delta immunoreactivity. cFLIP-delta was increased in a subset of reactive astrocytes in pericontusional cortex and hippocampus at 48 to 72 h. Low levels of both cFLIP isoforms were detected in human cortical tissue with no TBI, from four patients undergoing brain surgery for epilepsy and <24 h post mortem from three patients without CNS pathologic assessment. In cortical tissue surgically removed <18 h after severe TBI (n=3), cFLIP-alpha expression was increased relative to epilepsy controls (P<0.05) but not relative to post-mortem controls. The data suggest differential spatial and temporal regulation of cFLIP-alpha and cFLIP-delta expression that may influence the magnitude of cell death and further implicate programmed mechanisms of cell death after TBI.

Expression of cellular FLICE/caspase-8 inhibitory protein is associated with malignant potential in endometrial carcinoma

This study aimed to investigate the expression of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)/caspase-8 inhibitory protein (c-FLIP) in endometrial carcinoma and its possible implications. c-FLIP protein was detected in 42 endometrial carcinoma tissues and in 22 normal proliferative endometrial tissues by immunohistochemistry. In addition, c-FLIP messenger ribonucleic acid (mRNA) was evaluated in 20 endometrial carcinomas and in 18 normal proliferative endometria by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using SYBR Green I(TM). The relationship between c-FLIP protein level and tumor cell proliferation and that between c-FLIP protein level and clinicopathologic parameters of patients with endometrial carcinoma was analyzed. c-FLIP protein expression was significantly higher in neoplastic tissues than in normal tissues (P < 0.01), and similar result was obtained from RT-PCR analysis of c-FLIP mRNA (P < 0.01). Furthermore, c-FLIP protein was significantly associated with proliferating cell nuclear antigen-labeling index (P < 0.01), clinical stage (P < 0.05), the presence of invasion to > 1/2 myometrium (P < 0.05), and lymph node metastasis (P < 0.01). Multivariate analysis of variance also confirmed the association of c-FLIP with clinical stage (P < 0.05) and with lymph node metastasis (P < 0.05), while its association with myometrial invasion was marginal (P = 0.059). It is concluded that c-FLIP might contribute to the carcinogenesis and aggressiveness of endometrial carcinoma and might be a useful prognostic factor in the tumor.

Redox Regulation of Apoptosis by Members of the TNF Superfamily

Tumor necrosis factor (TNF), fibroblast-associated cell surface (Fas) ligand, and TNF-related apoptosisinducing ligand (TRAIL), all members of the TNF superfamily, are arguably the most potent inducers of cell death. These cytokines induce cell death through sequential recruitment by the death receptors TNFR1- associated death domain protein (TRADD), Fas-associated death domain protein (FADD), FADD-like interleukin-1beta-converting enzyme (FLICE), and downstream caspases. Increasing evidence indicates that mitochondria play a critical role in cytokine receptor-mediated apoptosis. There is also now ample evidence that apoptosis induced by TNF and its family members is mediated through the production of reactive oxygen intermediates (also known as reactive oxygen species). Here we review the evidence linking reactive oxygen intermediates to cytokine-induced cell death mediated by TNF-alpha/beta, Fas, TRAIL, TNF-like weak inducer of apoptosis (TWEAK), and vascular endothelial cell growth inhibitor (VEGI).

Pseudomonas aeruginosa Exotoxin A Induces Human Mast Cell Apoptosis by a Caspase-8 and -3-dependent Mechanism

Mast cells play an important role in both allergy and innate immunity. Recently, we demonstrated an active interaction between human mast cells and Pseudomonas aeruginosa leading to the production of multiple cytokines. Here, we show that both primary cultured human cord blood-derived mast cells and the human mast cell line HMC-1 undergo apoptosis as determined by single-stranded DNA (ssDNA) formation after stimulation with P. aeruginosa exotoxin A (ETA), a major toxin produced by this bacterium. ETA-induced ssDNA formation was completely inhibited by Z-VAD (where Z is benzyloxycarbonyl), which blocks multiple caspases, suggesting a role for caspases in this process. Active caspase-3 formation in mast cells after an ETA challenge was detected by both Western blotting and flow cytometry analysis. ETA-induced caspase-3 activity in human mast cells was demonstrated by the detection of a characteristic 23 kDa product of D4-GDI (where GDI is guanine nucleotide dissociation inhibitor), an endogenous caspase-3 substrate. Interestingly, a specific caspase-8 inhibitor, Z-IETD-fmk (where fmk is fluoromethyl ketone), blocked ETA-induced cleavage of D4-GDI, but a caspase-9 inhibitor (Z-LEHD-fmk) did not. Treatment of mast cells with caspase-3 inhibitor Z-DEVD-fmk or caspase-8 inhibitor Z-IETD-fmk reduced the generation of ssDNA induced by ETA, suggesting a role for caspase-8 and -3 in ETA-induced mast cell apoptosis. Furthermore, treatment of mast cells with ETA induced decreases of the short form and a long form (p43) of Fas-associated death domain protein (FADD)-like interleukin-1beta-converting enzyme (FLICE) (caspase-8)-inhibitory proteins (FLIPs), which are endogenous caspase-8 inhibitors. Taken together, these results suggest that ETA-induced mast cell apoptosis involves down-regulation of antiapoptotic proteins, FLIPs, and activation of caspase-8 and -3 pathways.

Possible role of FLICE-like inhibitory protein (FLIP) in chemoresistant ovarian cancer cells in vitro.

Chemoresistance is a major therapeutic problem and the current knowledge on cellular mechanisms involved is incomplete. In the present study, we have investigated the possible involvement of Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP) in ovarian cancer resistance by comparing chemosensitive (OV2008) and chemoresistant (C13*) ovarian cancer cells treated with cisplatin in vitro, and/or transfected with FLIP sense cDNA or FLIP small interfering RNA (siRNA) and determining FLIP protein content, cleavage of caspase-8 and caspase-3 and apoptosis. Cisplatin significantly decreased FLIP protein level, induced cleavage of caspase-8 and caspase-3 and apoptosis in a concentration-dependent manner in cisplatin-sensitive but not -resistant cells. While overexpression of FLIP-attenuated cisplatin-induced cleavage of caspase-8 and caspase-3 and apoptosis in chemosensitive cells, downregulation of FLIP in chemoresistant cells by siRNA increased apoptosis induced by cisplatin. These results suggest that FLIP plays a significant role in the regulation of apoptosis in human ovarian cancer cells and their sensitivity to cisplatin. This cell survival factor may be an important determinant in chemoresistance in ovarian cancer and may serve as a molecular target for the development of novel therapy for chemoresistant ovarian cancer.

Impairment of death-inducing signalling complex formation in CD95-resistant human primary lymphoma B cells.

Multiple mechanisms exist by which tumour cells can escape CD95-mediated apoptosis. Previous studies by our laboratory have shown that primary B cells from non-Hodgkin's Lymphoma (B-NHL) were resistant to CD95-induced cell death. In the current study, we have analysed the mechanisms underlying CD95 resistance in primary human lymphoma B cells. We report that FADD (FAS-associated death domain protein) and caspase-8 were constitutively expressed in lymphoma B cells and that the CD95 pathway was blocked upstream to caspase-8 activation. However, caspase-8 was processed and functional after treatment with staurosporine (STS). We found that the expression levels of FLICE (FADD-like interleukin-1 beta-converting enzyme)-Inhibitory Protein (c-FLIP) and Bcl-2-related proteins were heterogeneous in B-NHL cells and were not related to CD95 resistance. Finally, we report the absence of a CD95-induced signalling complex [death-inducing signalling complex (DISC)] in lymphoma B cells, with no FADD and caspase-8 recruitment to CD95 receptor. In contrast, DISC formation was observed in CD95-resistant non-tumoural (NT) B cells. Therefore, we propose that the absence of DISC formation in primary lymphoma B cells may contribute to protect these cells from CD95-induced apoptosis.

Regulation of TRAIL-Induced Apoptosis by Ectopic Expression of Antiapoptotic Factors

The discovery of an agent that selectively kills tumor cells and not normal cells is the dream of every cancer researcher. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), first discovered in 1995, was heralded as a selective killer of tumor cells, and its potential is still thought to be high. Almost immediately, broad efforts were made to understand its activity at the molecular level. TRAIL has been shown to interact with the cell surface through five distinct receptors, named death receptor (DR) 4, DR5, decoy receptor (Dc)R1, DcR2, and osteoprotegrin. It activates nuclear factor (NF)-kappaB, c-Jun N-terminal kinases, and apoptosis. The apoptotic signals are mediated through Fas-associated death domain protein (FADD)-mediated recruitment of caspase-8 and caspase-3. Additionally, caspase-8 can cleave Bcl-2 homology domain 3 (BH3)-interfering domain death agonist (Bid), and the cleaved Bid then causes the release of mitochondrial cytochrome c, leading to the activation of pro-caspase-9, which can then activate pro-caspase-3. TRAIL-induced apoptosis is negatively regulated by numerous cellular factors including decoy receptors, cellular FADD-like interleukin 1 beta-converting enzyme (FLICE) interacting protein (cFLIP), cellular inhibitor of apoptosis protein (cIAP), X-linked IAP (XIAP), survivin, and NF-kappaB. Second mitochondria-derived activator of caspases (Smac)?direct IAP binding protein with low pI (DIABLO) mediates proapoptotic signals through inaction of IAP. How the TRAIL-induced apoptosis is downregulated by these factors is discussed in detail in this review. Whether TRAIL selectively kills tumor cells without harming normal cells is also discussed.

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.

The induction of the TNFalpha death domain signaling pathway in Alzheimer's disease brain.

The tumor necrosis factor-alpha death domain pathway contributes to cellular degeneration in a variety of conditions. This study investigates the hypothesis that this death domain pathway is progressively induced in the brain during the progression of Alzheimer's disease (AD). AD cases had increased levels of proapoptotic markers including tumor necrosis factor-alpha (TNFalpha), TNF receptor type 1 (TNF-R1), TNF receptor-associated death domain (TRADD), and caspase-3, 2- to 10-fold higher (P < .01) than age-matched controls and 1 to 3 times higher than transitional cases. In striking contrast, potentially neuroprotective TNF receptor type 2 (TNF-R2), and Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE) inhibitor protein (FLIP) were decreased in AD as compared with age-matched control cases (P < .01). Overall, there was an elevation in proapoptotic elements, including a 5-fold increase in TNF-R1 and a 12-fold decrease in FLIP in AD brains. These changes may translate to increased degenerative potential because the downstream effector caspase-3 and product of the TNF pathway was also increased in parallel with enhanced TNF proapoptotic conditions. Our findings suggest that the TNF death receptor pathway and caspases are activated in the early stages of neuronal degeneration in AD.

Calcium/Calmodulin-dependent Protein Kinase II Regulation of c-FLIP Expression and Phosphorylation in Modulation of Fas-mediated Signaling in Malignant Glioma Cells

Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet also proliferative signals, which have been implicated in tumor pathogenesis. In this study, we investigated the molecular mechanisms that control Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11, induced apoptosis in sensitive glioma cells through caspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC) where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE)-inhibitory protein) to the Fas-mediated DISC. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells. Calcium/calmodulin-dependent protein kinase II (CaMK II) activity was up-regulated in resistant cells. Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II cDNA in sensitive cells rendered them resistant to CH-11. These results indicated that CaMK II regulates c-FLIP expression and phosphorylation, thus modulating Fas-mediated signaling in glioma cells.

A defect in central tolerance in NOD mice

The predisposition of nonobese diabetic (NOD) mice to develop autoimmune disease is usually attributed to defects in peripheral tolerance mechanisms. Here, evidence is presented that NOD mice display a defect in central tolerance (negative selection) of thymocytes. Impaired central tolerance in NOD mice was most prominent in a population of semi-mature thymocytes found in the medulla. The defect was apparent in vivo as well as in vitro, was independent of IAbetag7 expression and affected both Fas-dependent and Fas-independent pathways of apoptosis; for Fas-dependent apoptosis, the defective tolerance of NOD thymocytes correlated with the strong T cell receptor-mediated up-regulation of caspase 8-homologous FLICE (Fas-associated death-domain-like interleukin 1beta-converting enzyme)-inhibitory protein. In light of these findings, disease onset in NOD mice may reflect defects in central as well as peripheral tolerance.

Differential Display Identification of 40 Genes with Altered Expression in Activated Human Smooth Muscle Cells: LOCAL EXPRESSION IN ATHEROSCLEROTIC LESIONS OF smags, SMOOTH MUSCLE ACTIVATION-SPECIFIC GENES

Detailed knowledge on the molecular and cellular mechanisms that control (re)-differentiation of vascular smooth muscle cells (SMCs) is critical to understanding the pathological processes underlying atherogenesis. We identified by differential display/reverse transcriptase-polymerase chain reaction 40 genes with altered expression in cultured SMCs upon stimulation with the conditioned medium of activated macrophages. This set of genes comprises 10 known genes and 30 novel genes, which we call "smags" (for smooth muscle activation-specific genes). To determine the in vivo significance of these (novel) genes in atherogenesis, we performed in situ hybridization experiments on vascular tissue. Specifically, FLICE (Fas-associated death domain-like interleukin-1beta-converting enzyme)-like inhibitory protein (FLIP) is expressed in neointimal SMCs as well as in lesion macrophages and endothelial cells, whereas the expression of the novel genes smag-63, smag-64, and smag-84 is restricted to neointimal SMCs. Characterization of full-length smag-64 cDNA revealed that it encodes a novel protein of 66 amino acids. smag-82 cDNA comprises the complete, unknown, 3'-untranslated region of fibroblast growth factor-5. Collectively, our results illustrate the complex changes of SMC gene expression that occur in response to stimulation with cytokines and growth factors secreted by activated macrophages. Moreover, we identified interesting candidate genes that may play a role in the differentiation of SMCs during atherogenesis.

Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic beta cells.

Cytokines induce apoptosis in pancreatic beta cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-alpha) in the apoptosis of beta cells. Using the ribonuclease (RNase) protection assay and the reverse transcriptase-polymerase chain reaction (RT-PCR) method, we confirmed that TNF receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD), and FADD-like interleukin-1beta-converting enzyme (FLICE) were expressed in the pancreatic beta cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-alpha generated new 3'-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-alpha produced characteristic apoptotic patterns of DNA fragments formed by internucleosomal hydrolysis of static chromatin. In addition, C2-ceramides and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characteristic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. We also determined endosomal ceramide production after TNF-alpha (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-alpha can cause apoptosis in pancreatic beta cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.

Expression of K13/v-FLIP Gene of Human Herpesvirus 8 and Apoptosis in Kaposi's Sarcoma Spindle Cells

Human herpesvirus 8 (HHV8) infection is associated with all forms of Kaposi's sarcoma (KS). The HHV8 genome locus ORFK13-72-73 (ORF = open reading frame) encodes proteins that may be important in HHV8-mediated pathogenesis, i.e., the latency-associated nuclear antigen (encoded by ORF73), viral-cyc-D (v-cyc-D), a viral homologue of cellular cyclin D (encoded by ORF72), and viral-FLIP (v-FLIP), a homologue of the cellular FLICE (Fas-associated death domain-like interleukin 1 beta-converting enzyme) inhibitory protein (encoded by ORFK13; is an inhibitor of apoptosis [programmed cell death]). Through differential splicing events, this locus expresses individual RNA transcripts that encode all three proteins (tricistronic transcripts) or just two of them (v-FLIP and v-cyc-D; bicistronic transcripts). We examined expression of these transcripts in KS tissues. We collected tissues from patients with KS of different stages. By use of an optimized in situ hybridization procedure, we examined different ORFK13-72-73 locus transcripts in HHV8-infected cells in skin lesions and in one adjacent lymph node. Apoptosis in KS lesions was determined by use of an in situ assay. Our results indicate the following: 1) Transcripts from the ORFK13-72-73 locus appear to be spliced differentially in latently infected KS cells in skin lesions and in HHV8-infected cells in lymph nodes; specifically, ORFK13-ORF72 bicistronic transcripts were expressed abundantly in KS cells, whereas ORFK13-ORF72-ORF73 tricistronic transcripts were detected only in lymph node cells. 2) Sequences encoding the antiapoptotic protein v-FLIP are expressed at very low levels in early KS lesions, but expression increases dramatically in late-stage lesions. 3) The increase in expression of v-FLIP-encoding transcripts is associated with a reduction in apoptosis in KS lesions. These data suggest that functional v-FLIP is produced in vivo and that antiapoptotic mechanisms may be involved in the rapid growth of KS lesions, where only a few cells undergoing mitosis are generally observed.