Potentiation of Fas-mediated apoptosis by an engineered glycosylphosphatidylinositol-linked Fas

FasL and TRAIL are apoptotic ligands of the TNF-like cytokines family, acting via activation of the transmembrane death domain containing receptors Fas for FasL, and DR4 or DR5 for TRAIL. A glycosylphosphatidylinositol-linked TRAIL receptor called DcR1 behaves as a decoy receptor inhibiting TRAIL-mediated cell death in several cellular systems. We engineered and stably expressed a chimeric GPI-linked Fas receptor (Fas-GPI) in T-lymphocyte cell lines constitutively expressing functional transmembrane Fas. Surprisingly, despite lacking the death domain region of functional Fas, Fas-GPI was able to significantly increase Fas-mediated cell death triggered by membrane bound or soluble FasL, whereas engagement of Fas-GPI alone did not trigger apoptosis. This potentiating effect, but not transmembrane Fas activation, was selectively inhibited by protein kinase C activation with phorbol esters, demonstrating that Fas-GPI activated a specific synergistic signal transduction pathway. Fas-GPI and transmembrane Fas were localized in distinct membrane compartments, since Fas-GPI, but not transmembrane Fas, was found in the glycolipid-rich membrane microdomains. These results suggest that apoptosis induced by members of this ligand/receptors family may be differentially modulated through other and parallel signalling pathways.     

Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression

Phosphatidylserine (PS) exposure on the surface of cells has been considered a characteristic feature of apoptosis. However, we demonstrate herein that externalization of PS occurs in a cell-type-specific, albeit caspase-dependent, manner. Moreover, we could find no correlation in six different cell lines between the level of expression of the phospholipid (PL) scramblase and the capacity of these cells to externalize PS during apoptosis. Overexpression of PL scramblase in Raji cells, which exhibit low constitutive expression of this enzyme, by retroviral transduction of PL scramblase or treatment of the cells with interferon-alpha, failed to confer the capacity to expose PS in response to apoptotic stimuli. However, the lack of PS exposure in some cell types was not due to their inability to translocate PS molecules to the cell surface, since incubation with thiol reactive agents, such as N-ethylmaleimide, disulfiram and diamide, yielded rapid and pronounced PS exposure in all cell lines. These data suggest that plasma membrane PS exposure is not an obligatory component of the apoptotic phenotype, and that PL scramblase is not the sole determinant of PS externalization in apoptotic cells when this occurs.     

Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells

The expression of genes that regulate Fas-induced apoptosis has been examined in 10 human cultured colon carcinoma cell lines with defined and varied sensitivity to the cytolytic anti-Fas MoAb CH-11. Four lines demonstrated sensitivity to CH-11 (HT29, GC3/c1, TS-, Thy4), and six were resistant to the induction of apoptosis vis Fas. In nine lines expressing Fas, PCR-sequencing indicated that the death domain contained wt sequences. Downstream of Fas, expression of FADD/MORT1 and FLICE, essential components of the DISC, and negative regulators of Fas signalling including sFas, FAP-1 and Bcl-2, showed no correlation between levels of expression and sensitivity to Fas-mediated cytotoxicity. However, levels of the Fas antigen varied by >1000-fold, and correlated with CH-11 sensitivity. Following fourfold elevation in Fas expression in HT29 cells treated with interferon-gamma, a synergistic effect on Fas-mediated apoptosis was obtained when CH-11 and interferon-gamma were combined.     

Expression of caspase-3 and -7 does not correlate with the extent of apoptosis in primary breast carcinomas

Association between the rate of apoptosis and expression of the several relevant molecules (Bcl-2, pro- and active caspase-3, and caspase-7) was studied in 61 primary breast carcinomas. The rate of apoptosis detected both morphologically and by the TUNEL assay appeared to be high in 18 (30%), moderate in 14 (23%), and low in 29 (48%) carcinomas. High apoptotic index was strongly associated with advanced tumor grade and estrogen receptor positive (ER+) status but not with other investigated clinical or morphological parameters. Among the molecules studied, only the Bcl-2 protein expression demonstrated strong (inverse) correlation with the apoptotic index (p = 0.032). The data of this expected correlation was served as internal control in the study. Interestingly, high levels of the anti-apoptotic protein Bcl-2 was frequently co-incident with increased expression of pro-apoptotic molecules, such as active caspase-3 (p = 0.004) and caspase-7 (p = 0.001). However, expression of caspase-3 or caspase-7 did not show correlation with the extent of apoptosis or any clinico-morphological features, except overrepresentation of ER+ status in tumors expressing caspase-3 (p = 0.009). Thus, these findings indicate a general dysregulation of spontaneous apoptosis in primary breast tumors.     

Fas ligand-induced c-Jun kinase activation in lymphoid cells requires extensive receptor aggregation but is independent of DAXX, and Fas-mediated cell death does not involve DAXX, RIP, or RAIDD

Jun kinase signaling can be elicited by death receptor activation, but the mechanism and significance of this event are still unclear. It has been reported that cross-linking Abs to Fas trigger c-Jun N-terminal kinase (JNK) signaling via caspase-mediated activation of MEKK1 (JNK kinase kinase), elevation of ceramide levels or by recruitment of death domain associated protein (DAXX) to Fas. The effect of physiological ligand for Fas on JNK signaling was never investigated, although evidence is accumulating that Fas ligand is able to induce cellular responses distinct from those evoked by Ab-mediated cross-linking of Fas. Therefore, we investigated the effect of Fas ligand on JNK signaling. Like its ability to induce cell death, Fas ligand reliably activated JNK only upon extensive aggregation of the receptor. Although this was partially dependent on caspase activation, DAXX was not required. DAXX and other death receptor-associated proteins, which have been reported to bind directly or indirectly to Fas, such as receptor interacting protein (RIP) and RIP-associated ICH-1/CED-3-homologous protein with a death domain (RAIDD), were shown to be dispensable for Fas ligand-induced apoptosis.     

Steroid hormone toxicity in human fibroblasts does not correlate with high affinity receptor content.

Human diploid skin fibroblasts derived from normal individuals and those with the testicular feminization syndrome (TFM) have been shown to be killed to the same degree by dihydrotestosterone in spite of the absence of high affinity cellular androgen receptors in the TFM fibroblasts. Furthermore, several different normal fibroblast strains from various anatomical sites all showed similar amounts of androgen-induced cytotoxicity even though their respective receptor contents differed by as much as ten-fold. These results suggest that steroid-induced cytotoxicity in human fibroblasts is not correlated with receptor content, unlike murine lymphoid cells in which the receptor content has been shown to be closely related to their ability to survive hormone exposure.     

Proteases in Fas-mediated apoptosis

Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis. J. Cell. Biochem. 64:43–49. © 1997 Wiley-Liss, Inc.     

Apoptosis-linked gene 2 binds to the death domain of Fas and dissociates from Fas during Fas-mediated apoptosis in Jurkat cells

Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca(2+)-binding proteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation. We studied the regulation of ALG-2 during Fas-mediated apoptosis in Jurkat cells. The 22-kDa ALG-2 protein is cleaved and becomes a 19-kDa protein after Fas activation. The appearance of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/ml of anti-Fas Ab treatment and gradually degrades afterward. Confocal microscopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fas-mediated apoptosis. Therefore, we examined if ALG-2 interacts with Fas. The protein-protein interaction of ALG-2 with Fas was demonstrated using yeast two-hybrid assays as well as in vitro GST pull-down assay. Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation. However, the endogenous ALG-2 was no longer immunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment. This study, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new member of posttranslationally modified proteins during Fas-mediated apoptotic process.     

Soluble Fas gene therapy protects against Fas-mediated apoptosis of hepatocytes but not the lethal effects of Fas-induced TNF-alpha production by Kupffer cells

The elevation of soluble Fas (sFas) in the sera of patients with liver disease suggests a role for sFas in the disease process; whether it is protective or not is controversial. To determine the effects of sFas on Fas-induced liver apoptosis, we manipulated mice to produce sFas by transfecting them in vivo with different amounts of an adenovirus that produces mouse sFas driven by the CMV promoter (AdsFas). Fas-mediated apoptosis was induced by administration of anti-mouse Fas (Jo2; 10 microg/mouse) one week later. The administration of AdsFas (10(3), 10(7), or 10(9) pfu/mouse), which was associated with only minimal side-effects, resulted in a significant reduction in the liver transaminase levels and mortality of the mice on challenge with Jo2, as compared to control mice treated with AdLacZ. However, the protective effect of AdsFas was not complete. The possibility that Jo2-induction of TNF-alpha in the Kupffer cells of the liver contributes to the pathology was therefore tested. Although administration of soluble TNF receptor (sTNFRI) alone did not protect the mice from the lethal effects of Jo2, administration of sTNFRI (200 microg/mouse) after infection with AdsFas (10(9) pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-alpha by Kupffer cells produce the lethal effects of Jo2 that remained after treatment with AdsFas, these cells were selectively ablated by treatment of the mice with gadolinium chloride prior to challenge with Jo2. This treatment greatly reduced early mortality and hepatocellular damage as well as TNF-alpha production 6 h after injection of Jo2. These results indicate that: (1) AdsFas prevents Jo2-induced apoptosis of hepatocytes; (2) In addition to mediating Fas-mediated apoptosis of hepatocytes, Jo2 can separately induce TNF-alpha production by Kupffer cells resulting in early mortality, and (3) Optimal protection from Jo2-induced mortality can be achieved by protection of liver cells by pretreatment with both AdsFas and sTNFRI.     

Tissue factor expression and serum level in patients with melanoma does not correlate with disease progression.

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.     

Efficient delivery of siRNA into cytokine-stimulated insulinoma cells silences Fas expression and inhibits Fas-mediated apoptosis

Fas/FasL interactions have been proposed as a potentially important mechanism mediating beta-cell death in type 1 diabetes. Recent investigations suggest RNA interference, afforded by small interfering RNAs (siRNA), can provide specific and robust gene silencing in mammalian cells. The current study attempted to investigate the effects of silencing Fas expression with siRNA on Fas-mediated apoptosis in mouse insulinoma cells following cytokine incubation. Our results indicate that siRNA is capable of rapid inhibition of cytokine-induced Fas mRNA production and cell surface Fas protein. A complete suppression of the total Fas protein was only observed after prolonged incubation with siRNA, suggesting a slow turn-over of Fas protein. Moreover, siRNA significantly inhibited Fas-mediated beta-cell apoptosis assessed by Caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays, the extent of which positively correlated with the level of cell surface Fas. These observations provide additional evidence supporting a role for the Fas-mediated pathway in beta-cell destruction, and suggest that siRNA targeting Fas may be of therapeutic value in preventing type 1 diabetes and improving islet cell viability in transplantation.     

Thermal stability of proteins does not correlate with the energy of intramolecular interactions

Small monomeric proteins from mesophilic and thermophilic organisms were studied. They have close structural and physical and chemical properties but vary in thermal stability. A thermodynamic analysis of heat unfolding was made and integral enthalpy of unfolding (DeltaH(unf)), heat capacity of hydration (DeltaC(p)(hyd)) and enthalpy of hydration (DeltaH(hyd)) and of the buried surface area (DeltaASA) of nonpolar and polar groups as well as the enthalpy of disruption of intramolecular interaction (DeltaH(int) in gas phase) at 298 K were determined. The absence of correlation between protein thermostability and energetic components suggests that regulatory mechanism of protein thermal stabilization has entropic nature.     

1,2-Diacylglycerol accumulation in human neutrophils does not correlate with respiratory burst activation

Measurements of the level of 1,2-diacylglycerol (1,2-DG) during activation of the respiratory burst of human neutrophils by formyl-methionyl-leucyl-phenylalanine (fMLP) in the presence of platelet-activating factor (PAF) or by opsonized particles show that a correlation between accumulation of 1,2-DG and O2 consumption does not exist. Inhibition of protein kinase C activity with staurosporine before addition of opsonized particles demonstrates that the first phase of the respiratory burst is not inhibited, whereas the second phase, which is accompanied by a rise in the content of 1,2-DG, is strongly inhibited. This study indicates that accumulation of 1,2-DG cannot be the sole signal for the initiation of the respiratory burst in human neutrophils.     

Frequency of radiation-induced micronuclei in neuronal cells does not correlate with clonogenic survival

It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2alpha, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2alpha, G28, G120 and G44; SF2 >/= 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of (60)Co gamma radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2 相似文献   

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium. C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.     

Glutathione-induced radical formation on lactoperoxidase does not correlate with the enzyme's peroxidase activity

Lactoperoxidase (LPO) is believed to serve as a mediator of host defense against invading pathogens. The protein is more abundant in body fluids such as milk, saliva, and tears. Lactoperoxidase is known to mediate the oxidation of halides and (pseudo)halides in the presence of hydrogen peroxide to reactive intermediates presumably involved in pathogen killing. More recently, LPO has been shown to oxidize a wide diversity of thiol compounds to thiyl free radicals, which ultimately lead to the formation of a protein radical characterized by DMPO-immunospin trapping. In the same study by our group the authors claimed that a consequence of this protein radical formation was the inactivation of LPO (Guo et al., J. Biol. Chem.279:13272-13283; 2004). Here we demonstrate that although thiyl radical formation does lead to LPO radical production, the formation of this radical is unrelated to the enzyme's activity. We suggest the source of this misleading interpretation to be the binding of GSH to ELISA plates, which interferes with ABTS and guaiacol oxidation. In addition, DMPO-GSH-nitrone adducts bind to ELISA plates, leading to ambiguities of interpretation since we have demonstrated that DMPO-GSH nitrone does not bind to LPO, and only LPO-protein-DMPO-nitrone adducts can be detected by Western blot.     

Diphosphate concentration does not correlate with the level of inorganic diphosphatase inEscherichia coli

Partial inhibition of DNA synthesis stimulates the production of inorganic diphosphatase inEscherichia coli but the changes in diphosphate (PP _i) level observed did not correlate with the enzyme activity. An accumulation ofPP _I was observed in the presence of inhibitors of RNA synthesis or nucleotide synthesis. In the former case the level of the enzyme did not change but in the latter case it increased. Thus the amount of inorganic diphosphatase alone does not determine the concentration ofPP ₁ inE. coli.