FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation

Fas is a cell surface death receptor that signals apoptosis. Several proteins have been identified that bind to the cytoplasmic death domain of Fas. Fas-associated death domain (FADD), which couples Fas to procaspase-8, and Daxx, which couples Fas to the Jun NH(2)-terminal kinase pathway, bind independently to the Fas death domain. We have identified a 130-kD kinase designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fas-interacting protein. Binding to Fas is mediated by a conserved sequence in the COOH terminus of the protein. FIST/HIPK3 is widely expressed in mammalian tissues and is localized both in the nucleus and in the cytoplasm. In transfected cell lines, FIST/HIPK3 causes FADD phosphorylation, thereby promoting FIST/HIPK3-FADD-Fas interaction. Although Fas ligand-induced activation of Jun NH(2)-terminal kinase is impaired by overexpressed active FIST/HIPK3, cell death is not affected. These results suggest that Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas.     

Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis

Engagement of the Fas receptor (CD95) initiates multiple signaling pathways that lead to apoptosis, such as the formation of death-inducing signaling complex (DISC), activation of caspase cascades, and the generation of the lipid messenger, ceramide. Sphingomyelin (SM) is a major component of lipid rafts, which are specialized structures that enhance the efficiency of membrane receptor signaling and are a main source of ceramide. However, the functions of SM in Fas-mediated apoptosis have yet to be clearly defined, as the responsible genes have not been identified. After cloning a gene responsible for SM synthesis, SMS1, we established SM synthase-defective WR19L cells transfected with the human Fas gene (WR/Fas-SM(-)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas-SMS1). We show that expression of membrane SM enhances Fas-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of Fas into lipid rafts, and subsequent Fas clustering. Furthermore, WR/Fas-SMS1 cells, but not WR/Fas-SM(-) cells, showed a considerable increase in ceramide generation within lipid rafts upon Fas stimulation. These data suggest that a membrane SM is important for Fas clustering through aggregation of lipid rafts, leading to Fas-mediated apoptosis.     

Mechanism of action of anti-proliferative lysophospholipid analogues against the protozoan parasite Trypanosoma cruzi: potentiation of in vitro activity by the sterol biosynthesis inhibitor ketoconazole

We investigated the mechanism of action of metabolically stable lysophospholipid analogues (LPAs), with potent anti-tumour and anti-protozoal activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Against the axenically grown epimastigote form of the parasite, the IC(50)s after 120 h for ET-18-OCH(3), miltefosine and ilmofosine were 3, 1 and 3 microM, respectively; at higher concentrations immediate lytic effects were observed. Eradication of the intracellular amastigote, grown inside Vero cells, was achieved at 0.1, 0.1 and 1 microM for ET-18-OCH(3), miltefosine and ilmofosine, respectively. Analysis of the lipid composition of epimastigotes exposed to LPAs at their IC(50) for 120 h showed that the ratio of phosphatidyl-choline (PC) to phosphatidylethanolamine (PE) changed from 1.5 in control cells to c. 0.67 in those treated with the analogues. A significant increase in the content of phosphatidylserine was also observed in treated cells. Intact epimastigotes efficiently incorporated radioactivity from L-[methyl-(14)C]methionine into PC, but not from [methyl-(14)C]choline. ET-18-OCH(3) inhibited the incorporation of L-[methyl-(14)C]methionine into PC with an IC(50) of 2 microM, suggesting that inhibition of the de novo synthesis through the Greenberg's pathway was a primary effect underlying the selective anti-parasitic activity of this compound. Antiproliferative synergism was observed as a consequence of combined treatment of epimastigotes with ET-18-OCH(3) and ketoconazole, a sterol biosynthesis inhibitor, probably due to the fact that a secondary effect of the latter is also a blockade of PC synthesis at the level of PE-PC-N-methyl-transferase.     

Interactions between alkylglycerols and human neutrophil granulocytes

We evaluated whether various alkylglycerols would initiate a functional response of human neutrophils or modify responses induced by a formyl peptide (fMLP) in vitro. We found that platelet activating factor (PAF) was the most potent with regard to the ability to produce an oxidative response (assessed by cytochrome c reduction and/or chemiluminescence), followed by ET-16-OCH3. Lyso-PAF, ET-18-OCH3, batyl- and chimyl alcohols exhibited only a weak activity. PAF was also the most efficient lipid conferring a rise of intracellular calcium concentrations ([Ca2+]i). ET-16-OCH3, ET-18-OCH3 and lysoPAF were less potent, although maximal [Ca2+]i levels were similar to that of 0.1 mumol/l fMLP. The kinetics of the calcium responses were highly specific for each ether lipid. When neutrophils had been treated with PAF or ET-18-OCH3 and were subsequently stimulated by fMLP, enhancement of the oxidative response was noted. Thus, this study shows that there was an association between the ability of an alkylglycerol to initiate oxidative and calcium responses, indicating strict structure-activity relationships for these lipids.     

High level expression of CD43 inhibits T cell receptor/CD3-mediated apoptosis

In a screen designed to identify genes that regulate T cell receptor (TCR)/CD3-mediated apoptosis, we found that high level expression of CD43 protected T cell hybridomas from activation-induced cell death. The protection appears to result from its capacity to block Fas-mediated death signals rather than from inhibition of the upregulation of Fas and/or Fas ligand after T cell stimulation. We found that peripheral CD4(+) T cells can be divided into two subsets based on the level of CD43 surface expression. The CD4(+)CD43(low) subset exhibits a naive T cell phenotype, being CD62L(high)CD45RB(high)CD44(low), whereas CD4(+)CD43(high) cells exhibit a memory phenotype, being CD62L(low)CD45RB(low)CD44(high). Recent studies have demonstrated that engagement of TCR and Fas induces naive CD4(+) T cells to undergo apoptosis, and the same treatment enhances the proliferation of memory CD4(+) T cells. We confirm here that peripheral CD4(+)CD43(high) T cells are resistant to TCR/CD3-mediated cell death. These results suggest that the expression levels of CD43 on naive and memory CD4(+) T cells determine their susceptibility to Fas-dependent cell death and that high level expression of CD43 may be used as a marker to define CD4(+) memory T cells. Expression of CD43 provides a novel mechanism by which tumor cells expressing abnormally high levels of CD43 may escape Fas-mediated killing.     

Dual Signaling of the Fas Receptor: Initiation of Both Apoptotic and Necrotic Cell Death Pathways

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, tetrapeptide inhibitors of caspase-1– and caspase-3–like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas–induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor κB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.     

Determination of the alkyl lysophospholipid derivative ET-18-OCH3, a new antineoplastic drug, in plasma

We describe a sensitive method for measuring the concentration of the new antineoplastic drug ET-18-OCH3 in plasma. After plasma lipids are extracted, ET-18-OCH3 is separated from the excess of endogenous lipids by thin-layer chromatography and specific enzymatic hydrolysis of sphingomyelin by the action of sphingomyelinase. Analytical recovery after the complete isolation was 73.5% (CV = 9.8%, n = 15). [3H]-ET-18-OCH3 is used as internal standard. A densitometric method in which 8-anilino-1-naphthalenesulfonate, Mg salt, is used as fluorescent agent (excitation at 367 nm and emission greater than 390 nm) allows the sensitive determination of ET-18-OCH3 down to 0.1 mg/L (CV greater than 30%). The day-to-day CV is 25% for concentrations of 0.15 to 0.625 mg/L, 12% for 1.5 to 5.0 mg/L. Preliminary pharmacokinetic data reveal gastrointestinal absorption of ET-18-OCH3 after multiple oral administration.     

The machinery of programmed cell death.

Apoptosis or programmed cell death is an essential physiological process that plays a critical role in development and tissue homeostasis. However, apoptosis is also involved in a wide range of pathological conditions. Apoptotic cells may be characterized by specific morphological and biochemical changes, including cell shrinkage, chromatin condensation, and internucleosomal cleavage of genomic DNA. At the molecular level, apoptosis is tightly regulated and is mainly orchestrated by the activation of the aspartate-specific cysteine protease (caspase) cascade. There are two main pathways leading to the activation of caspases. The first of these depends upon the participation of mitochondria (receptor-independent) and the second involves the interaction of a death receptor with its ligand. Pro- and anti-apoptotic members of the Bcl-2 family regulate the mitochondrial pathway. Cellular stress induces pro-apoptotic Bcl-2 family members to translocate from the cytosol to the mitochondria, where they induce the release of cytochrome c, while the anti-apoptotic Bcl-2 proteins work to prevent cytochrome c release from mitochondria, and thereby preserve cell survival. Once in the cytoplasm, cytochrome c catalyzes the oligomerization of apoptotic protease activating factor-1, thereby promoting the activation of procaspase-9, which then activates procaspase-3. Alternatively, ligation of death receptors, like the tumor necrosis factor receptor-1 and the Fas receptor, causes the activation of procaspase-8. The mature caspase may now either directly activate procaspase-3 or cleave the pro-apoptotic Bcl-2 homology 3-only protein Bid, which then subsequently induces cytochrome c release. Nevertheless, the end result of either pathway is caspase activation and the cleavage of specific cellular substrates, resulting in the morphological and biochemical changes associated with the apoptotic phenotype.     

Improved artificial death switches based on caspases and FADD.

A number of "suicide genes" have been developed as safety switches for gene therapy vectors or as potential inducible cytotoxic agents for hyperproliferative disorders, such as cancer or restenosis. However, most of these approaches have relied on foreign proteins, such as HSV thymidine kinase, that primarily target rapidly dividing cells. In contrast, novel artificial death switches based on chemical inducers of dimerization (CIDs) and endogenous proapoptotic molecules function efficiently in both dividing and nondividing cells. In this approach, lipid-permeable, nontoxic CIDs are used to conditionally cross-link target proteins that are fused to CID-binding domains (CBDs), thus activating signaling cascades leading to apoptosis. In previous reports, CID-regulated Fas and caspases 1, 3, 8, and 9 were described. Since the maximum efficacy of these artificial death switches requires low basal and high specific activity, we have optimized these death switches for three parameters: (1) extent of oligomerization, (2) spacing between CBDs and target proteins, and (3) intracellular localization. We describe improved conditional Fas and caspase 1, 3, 8, and 9 alleles that function at subnanomolar levels of the CID AP1903 to trigger apoptosis. Further, we demonstrate for the first time that oligomerization of the death effector domain of the Fas-associated protein, FADD, is sufficient to trigger apoptosis, suggesting that the primary function of FADD, like that of Apaf-1, is oligomerization of associated caspases. Finally, we demonstrate that nuclear-targeted caspases 1, 3, and 8 can trigger apoptosis efficiently, implying that the cleavage of nuclear targets is sufficient for apoptosis.     

Up-regulation of reactive oxygen species (ROS) and resistance to Fas-mediated apoptosis in the C33A cervical cancer cell line transfected with IL-18 receptor.

Cervical cancer cells were transfected with a newly discovered interleukin (IL)-18 receptor to investigate the effect of endogenous IL-18 on the regulation of immune-related factors such as Fas (CD95/Apo-1)/Fas ligand and intercellular adhesion molecules. Transfection of the IL-18 receptor selectively induced a slight enhancement of the Fas via the up-regulation of intracellular reactive oxygen species and IL-18 in cervical carcinoma C33A cells, whereas there were no effects on the expression of p53, intercellular adhesion molecules-1 and Fas ligand. Neither IL-18 receptor transfection nor recombinant IL-18 enhanced interferon-gamma production in C33A cells. Thus, IL-18 receptor transfection induced IL-18 expression and enhanced intracellular reactive oxygen species and Fas expression in C33A cells in an interferon-gamma-independent pathway. However, treatment with agonistic anti-Fas antibody did not induce the apoptosis of C33A/IL-18 receptor transfectants, suggesting that either reactive oxygen species play a key role in resisting the Fas-induced apoptosis of C33A cells, or Fas was not functional. These results show that C33A/IL-18 receptor cells are resistant to the apoptosis and thus can survive against the immune surveillance and activated immune cells. Our results thus suggest that IL-18 and IL-18 receptor, together, may play a role in immunoregulation or in inflammation by augmenting the levels of IL-18 and reactive oxygen species in C33A cells.     

Signaling and regulatory mechanisms of integrinalpha3beta1 on the apoptosis of cultured rat podocytes

Integrin is the major adhesion molecule for the attachment of podocytes to the glomerular basement membrane, and integrins have been shown to play a major role in the regulation of cell survival. In this study, the authors investigated the apoptosis and its related signal pathways to integrin in cultured rat podocytes. Apoptosis was detected with the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. Cytochrome c was examined by immunohistochemical stain, and Fas, Fas ligand, Bax, Bcl-2, and ERK activation (p-ERK/ERK) were analyzed by Western blotting analysis. The results demonstrated that the integrin antagonist, Gly-Arg-Gly-Asp (GRGD), increased the percentage of cells with apoptosis (from 0.9+/-0.5% to 27.2+/-9.9%, P < 0.01). Inhibition of protein tyrosine kinase with genistein also caused apoptosis of podocytes (from 0.9+/-0.5% to 26.0+/-8.7%, P < 0.01). In GRGD-treated cells, cytochrome c was found released into cytoplasm by immunohistochemical study and the Bax expression was upregulated, whereas Bcl-2 expression was not changed. Fas was not expressed in both control and GRGD-treated podocytes, although Fas ligand was upregulated in GRGD-treated cells. ERK activation was also found to be increased in GRGD-treated cells. The results indicated that alpha3beta1integrin is necessary for the prevention of the apoptosis of cultured rat podocytes, and that the signaling involves the Bax, Bcl-2, and cytochrome c pathways.     

T Cell Receptor Signals Enhance Susceptibility to Fas-mediated Apoptosis

Fas(CD95) and its ligand (FasL) interaction plays a pivotal role in T cell receptor (TCR)-mediated apoptosis. However, the susceptibility of T cells to Fas-mediated apoptosis is tightly regulated during immune responses, a regulation which is thought to maintain the antigen-specificity of T cell apoptosis. Here we show that TCR stimulation enhances the induction of Fas-mediated apoptosis. In addition, using a mutant T cell hybridoma with impaired FasL expression, we show that the synergy provided by TCR stimulation can be mimicked by activators of PKC but not calcium influx. This effect cannot be inhibited by actinomycin D, suggesting that TCR stimulation leads to the alteration in preexisting signaling molecules to enhance Fas-mediated apoptosis. Our results therefore provide a mechanism of how Fas-FasL interactions lead to T cell death in an antigen-specific manner via repetitive antigen stimulation.     

The molecular basis for apoptotic defects in patients with CD95 (Fas/Apo-1) mutations

Heterozygous mutations of the receptor CD95 (Fas/Apo-1) are associated with defective lymphocyte apoptosis and a clinical disease characterized by lymphadenopathy, splenomegaly, and systemic autoimmunity. From our cohort of 11 families, we studied eight patients to define the mechanisms responsible for defective CD95-mediated apoptosis. Mutations in and around the death domain of CD95 had a dominant–negative effect that was explained by interference with the recruitment of the signal adapter protein, FADD, to the death domain. The intracellular domain (ICD) mutations were associated with a highly penetrant Canale-Smith syndrome (CSS) phenotype and an autosomal dominant inheritance pattern. In contrast, mutations affecting the CD95 extracellular domain (ECD) resulted in failure of extracellular expression of the mutant protein or impaired binding to CD95 ligand. They did not have a dominant–negative effect. In each of the families with an ECD mutation, only a single individual was affected. These observations were consistent with differing mechanisms of action and modes of inheritance of ICD and ECD mutations, suggesting that individuals with an ECD mutation may require additional defect(s) for expression of CSS.     

The c-FLIP-NH2 terminus (p22-FLIP) induces NF-kappaB activation

c-FLIP proteins (isoforms: c-FLIP(L), c-FLIP(S), and c-FLIP(R)) play an essential role in the regulation of death receptor-induced apoptosis. Here, we demonstrate that the cytoplasmic NH2-terminal procaspase-8 cleavage product of c-FLIP (p22-FLIP) found in nonapoptotic malignant cells, primary T and B cells, and mature dendritic cells (DCs) strongly induces nuclear factor kappaB (NF-kappaB) activity by interacting with the IkappaB kinase (IKK) complex via the IKKgamma subunit. Thus, in addition to inhibiting apoptosis by binding to the death-inducing signaling complex, our data demonstrate a novel mechanism by which c-FLIP controls NF-kappaB activation and life/death decisions in lymphocytes and DCs.     

A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells

Single-chain alkylphospholipids, unlike conventional chemotherapeutic drugs, act on cell membranes to induce apoptosis in tumor cells. We tested four different alkylphospholipids, i.e., edelfosine, perifosine, erucylphosphocholine, and compound D-21805, as inducers of apoptosis in the mouse lymphoma cell line S49. We compared their mechanism of cellular entry and their potency to induce apoptosis through inhibition of de novo biosynthesis of phosphatidylcholine at the endoplasmic reticulum. Alkylphospholipid potency closely correlated with the degree of phosphatidylcholine synthesis inhibition in the order edelfosine > D-21805 > erucylphosphocholine > perifosine. In all cases, exogenous lysophosphatidylcholine, an alternative source for cellular phosphatidylcholine production, could partly rescue cells from alkylphospholipid-induced apoptosis, suggesting that phosphatidylcholine biosynthesis is a direct target for apoptosis induction. Cellular uptake of each alkylphospholipid was dependent on lipid rafts because pretreatment of cells with the raft-disrupting agents, methyl-beta-cyclodextrin, filipin, or bacterial sphingomyelinase, reduced alkylphospholipid uptake and/or apoptosis induction and alleviated the inhibition of phosphatidylcholine synthesis. Uptake of all alkylphospholipids was inhibited by small interfering RNA (siRNA)-mediated blockage of sphingomyelin synthase (SMS1), which was previously shown to block raft-dependent endocytosis. Similar to edelfosine, perifosine accumulated in (isolated) lipid rafts independent on raft sphingomyelin content per se. However, perifosine was more susceptible than edelfosine to back-extraction by fatty acid-free serum albumin, suggesting a more peripheral location in the cell due to less effective internalization. Overall, our results suggest that lipid rafts are critical membrane portals for cellular entry of alkylphospholipids depending on SMS1 activity and, therefore, are potential targets for alkylphospholipid anticancer therapy.     

Mode of action and molecular target of ECH, a specific inhibitor of death receptor-dependent apoptosis

ECH (epoxycyclohexenone) specifically blocks death receptor-mediated apoptosis induced by anti-Fas antibody, Fas ligand, or TNF-alpha, whereas it has no effect on death receptor independent apoptosis induced by staurosporine, MG-132, C2-ceramide, or UV irradiation. ECH blocks the activation of pro-caspase-8 in the death-inducing signaling complex (DISC), even though recruitment of FADD and pro-caspase-8 is not affected. In Fas ligand treated cells, ECH is only able to inhibit the activation of pro-caspase-8 and it has no effect on the already-activated caspase-8. ECH has a relatively higher affinity to pro-caspase-8, although it directly binds both pro- and active-form of caspase-8. In conclusion, ECH targets pro-caspase-8 and blocks the self-activation of pro-caspase-8 in the DISC, and thus selectively inhibits death receptor-mediated apoptosis. Moreover novel non-peptide inhibitors, RKTS-33 & RKTS-34 that are chemically synthesized derivatives of ECH have been developed.     

1-O-octadecyl-2-O-methyl-glycerophosphocholine inhibits the transduction of growth signals via the MAPK cascade in cultured MCF-7 cells.

1-O-Octadecyl-2-O-methyl-glycerophosphocholine (ET18-OCH3) is an ether lipid with selective antiproliferative properties whose mechanism of action is still unresolved. We hypothesized that since ET18-OCH3 affects a wide variety of cells, its mechanism of action was likely to involve the inhibition of a common widely used pathway for transducing growth signals such as the mitogen-activated protein kinase (MAPK) cascade. To test this, we established conditions whereby quiescent MCF-7 cells took up ET18-OCH3 in sufficient quantities that inhibited cell proliferation subsequent to the addition of growth medium and examined the activation of components of the MAPK cascade under these conditions. ET18-OCH3 inhibited the sustained phosphorylation of MAPK resulting in a decrease in the magnitude and duration of activation of MAPK in cells stimulated with serum or EGF. ET18-OCH3 had no effect on the binding of EGF to its receptors, their activation, or p21ras activation. However, an interference in the association of Raf-1 with membranes and a resultant decrease in Raf-1 kinase activity in membranes of ET18-OCH3-treated cells was observed. ET18-OCH3 had no direct effect on MAPK or Raf-1 kinase activity. A direct correlation between ET18-OCH3 accumulation, inhibition of cell proliferation, Raf association with the membrane, and MAPK activation was also established. These results suggest that inhibition of the MAPK cascade by ET18-OCH3 as a result of its effect on Raf-1 activation may be an important mechanism by which ET18-OCH3 inhibits cell proliferation.     

Caspase activation is required for T cell proliferation

Triggering of Fas (CD95) by its ligand (FasL) rapidly induces cell death via recruitment of the adaptor protein Fas-associated death domain (FADD), resulting in activation of a caspase cascade. It was thus surprising that T lymphocytes deficient in FADD were reported recently to be not only resistant to FasL-mediated apoptosis, but also defective in their proliferative capacity. This finding suggested potentially dual roles of cell growth and death for Fas and possibly other death receptors. We report here that CD3-induced proliferation and interleukin 2 production by human T cells are blocked by inhibitors of caspase activity. This is paralleled by rapid cleavage of caspase-8 after CD3 stimulation, but no detectable processing of caspase-3 during the same interval. The caspase contribution to T cell activation may occur via TCR-mediated upregulation of FasL, as Fas-Fc blocked T cell proliferation, whereas soluble FasL augmented CD3-induced proliferation. These findings extend the role of death receptors to the promotion of T cell growth in a caspase-dependent manner.     

New disease-modifying antirheumatic drug 2 acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298) selectively augments activation-induced T cell death.

We examined in this study whether the newly developed disease-modifying antirheumatic drug (DMARD) 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298) augments activation-induced T cell death. Peripheral blood (PB) T cells, isolated from healthy donors, were activated by incubation with interleukin-2 (IL-2) followed by further culture with 12-0-tetradecanoyl phorbol 13-acetate (PMA) and ionomycin in the presence or absence of KE-298. The apoptosis of activated T cells was examined by flow cytometric determination of hypodiploid DNA. Fas expression and caspase-3 activity in activated T cells were also examined by flow cytometry, and expression of Fas ligand (FasL), Bcl-2-related proteins, and X chromosome-linked inhibitor of apoptosis protein (XIAP) was determined by Western blot analysis. Apoptosis was not obvious in resting T cells and was not augmented by KE-298. In contrast, apoptosis was clearly detected in activated T cells (activation-induced T cell death) with the increment of caspase-3 activity, and incubation of these cells with KE-298 further enhanced apoptosis. Treatment of activated T cells with KE-298 increased Bax expression but decreased XIAP expression without affecting the expression of Fas/FasL. Thus caspase-3 activity in activated T cells appeared to be increased by KE-298. Our results suggest that the newly developed DMARD, KE-298, selectively augmented activation-induced T cell death. This finding may contribute to the therapeutic efficacy of KE-298 in rheumatoid arthritis (RA) patients and provide new insight into the pharmacologic action of DMARDs.     

Fas死亡结构域调节白血病细胞Meg-01内c-myc的表达

目的 探讨人白血病Meg-01细胞的异常增殖机制以及凋亡因子Fas对细胞异常增殖的调节作用。方法 用基因重组技术将Fas的细胞外区、跨膜区与白血病抑制因子（LIF）受体两亚基（gp190,gp130)细胞内区构成嵌合型受体（Fas/190,Fas/130),同时,将Fas死亡区域（FASDD）替换Fas/130中gp130细胞内区无结构域部分（Fas/130f),分别在Meg-01细胞内表达后,用抗Fas抗体激活这些嵌合型受体,随后用免疫组化和免疫印迹法分析受体细胞内区形成同源性寡聚体（190cyt-190cyt-190cyt,130cyt-130cyt-130cyt及FASDD－FASDD－FASDD）后的细胞状况和细胞内c-myc的表达。结果 转染pED Fas/130后用抗Fas抗体作用6－8h,Meg-01细胞c-myc表达量增加,细胞增殖明显;而pED Fas/190形成寡聚体后,Meg-01细胞c-myc的表达有所下降。另外,Fas/130与Fas/130f比较,后者的c-myc的表达明显下降,细胞形态大小不一。结论 LIF受体中gp130亚基细胞内区促进白血病Meg-01细胞内的c-myc表达及细胞增殖信号的传递;Fas死亡域可抑制gp130的细胞增殖活性。