Critical role for tumor necrosis factor-related apoptosis-inducing ligand in immune surveillance against tumor development.

Natural killer (NK) cells and interferon (IFN)-gamma have been implicated in immune surveillance against tumor development. Here we show that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays a critical role in the NK cell-mediated and IFN-gamma-dependent tumor surveillance. Administration of neutralizing monoclonal antibody against TRAIL promoted tumor development in mice subcutaneously inoculated with a chemical carcinogen methylcholanthrene (MCA). This protective effect of TRAIL was at least partly mediated by NK cells and totally dependent on IFN-gamma. In the absence of TRAIL, NK cells, or IFN-gamma, TRAIL-sensitive sarcomas preferentially emerged in MCA-inoculated mice. Moreover, development of spontaneous tumors in p53(+/-) mice was also promoted by neutralization of TRAIL. These results indicated a substantial role of TRAIL as an effector molecule that eliminates developing tumors.     

Glycogen synthase kinase-3beta suppression eliminates tumor necrosis factor-related apoptosis-inducing ligand resistance in prostate cancer

Prostate cancer is a major health threat for American men. Therefore, the development of effective therapeutic options is an urgent issue for prostate cancer treatment. In this study, we evaluated the effect of glycogen synthase kinase-3beta (GSK-3beta) suppression on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human prostate cancer cell lines. In the presence of lithium chloride (LiCl) or SB216763, the GSK-3beta inhibitors, TRAIL-induced cell death was dramatically enhanced, and the enhanced cell death was an augmented apoptotic response evidenced by increased Annexin V labeling and caspase-3 activation. GSK-3beta gene silencing mediated by a small interference RNA (siRNA) duplex also sensitized the cells to TRAIL, confirming the specificity of GSK-3beta suppression. Importantly, TRAIL stimulation increased GSK-3beta tyrosine phosphorylation at Y216, suggesting that GSK-3beta is activated by TRAIL. Furthermore, TRAIL sensitization was associated with increased proteolytic procession of caspase-8 and its downstream target BID, and z-IETD-FMK, the inhibitor specific to active caspase-8 totally blocked LiCl-induced TRAIL sensitization. Finally, Trichodion, a potent nuclear factor-kappaB (NF-kappaB) inhibitor, could not affect LiCl-induced TRAIL sensitization, although GSK-3beta inhibitors significantly blocked TRAIL-reduced NF-kappaB activity in prostate cancer cells. These results indicate that GSK-3beta suppression sensitizes prostate cancer cells to TRAIL-induced apoptosis that is dependent on caspase-8 activities but independent of NF-kappaB activation, and suggest that a mechanism involving GSK-3beta activation may be responsible for TRAIL resistance in prostate cancer cells.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to interferon gamma-dependent natural killer cell protection from tumor metastasis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-gamma production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, alpha-galactosylceramide (alpha-GalCer), a powerful inducer of NKT cell IFN-gamma and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-gamma-deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and alpha-GalCer were strictly IFN-gamma dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-gamma-mediated antimetastatic effects of IL-12 and alpha-GalCer.     

Ingenol 3-angelate induces dual modes of cell death and differentially regulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in melanoma cells

Ingenol 3-angelate (PEP005), one of the active ingredients in an extract from Euphorbia peplus, was shown in preclinical studies to have activity against human melanoma xenografts in nude mice. In the present study, we have tested its ability to induce the apoptosis of melanoma cells in vitro in the absence or presence of tumor necrosis factor-related apoptosis inducing ligand (TRAIL). The results showed that at relatively high concentrations (100 microg/mL), PEP005 killed melanoma cells mainly by induction of necrosis. In 20% of cell lines, evidence of apoptosis was observed. Apoptosis was caspase-dependent and associated with changes in mitochondrial membrane potential that were not inhibitable by overexpression of Bcl-2 or inhibition of caspases but were blocked by inhibition of protein kinase C (PKC). Low concentrations (1 or 10 microg/mL) of PEP005 either increased or decreased TRAIL-induced apoptosis in a cell line-dependent manner. These changes in TRAIL-induced apoptosis seemed to be due to activation of PKC and varying levels of PKC isoenzymes in different melanoma cell lines. PEP005-mediated enhancement of apoptosis seemed to be associated with low expression of the PKCepsilon isoform. These results indicate that PEP005 may enhance or inhibit sensitivity of melanoma to treatments associated with TRAIL-induced apoptosis depending on the PKC isoform content of melanoma cells.     

肝细胞生长因子在肿瘤坏死因子相关凋亡诱导配体作用下对原代肝星状细胞增殖及凋亡的影响

背景:活化的肝星状细胞是肝纤维化的关键因素,研究表明肝细胞生长因子能促进星状细胞凋亡,其具体机制可能与增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导星状细胞凋亡有关。目的:观察肿瘤坏死因子相关凋亡诱导配体作用下,肝细胞生长因子对原代肝星状细胞增殖、凋亡的影响并初步探讨其可能机制。方法:将SD大鼠原代肝星状细胞复苏、传代,细胞增殖明显时用于实验。实验分为4组:空白对照组为单纯肝星状细胞培养;肝细胞生长因子组:将100μg/L肝细胞生长因子作用于肝星状细胞;TRAIL组:将2mg/L的TRAIL作用于肝星状细胞;肝细胞生长因子+TRAIL组:将肝细胞生长因子预先刺激肝星状细胞24h,再加入2mg/LTRAIL。结果与结论:MTT检测显示肝细胞生长因子及TRAIL分别在50～200μg/L、0.5～1.5mg/L各浓度下对肝星状细胞增殖抑制率无影响,TRAIL在2mg/L作用下对肝星状细胞有抑制作用。流式细胞仪检测肝细胞生长因子+TRAIL组的中晚期凋亡率明显高于空白对照组及肝细胞生长因子组(P<0.05);肝细胞生长因子+TRAIL组DR5荧光强度明显高于其他3组(P<0.01)。提示在TRAIL作用下,肝细胞生长因子能促进肝星状细胞的凋亡、抑制其增殖。可能与肝细胞生长因子上调活化肝星状细胞表面DR5表达有关。     

Endotoxin increases plasma soluble tumor necrosis factor-related apoptosis-inducing ligand level mediated by the p38 mitogen-activated protein kinase signaling pathway

Despite extensive knowledge about the mechanisms behind sepsis, this syndrome still caries a large morbidity and mortality rate. Dysregulated immune and coagulation systems are held responsible. However, additional pathophysiological mechanisms such as uncontrolled apoptosis induced by death receptor ligands might well play a role. P38 mitogen-activated protein (MAP) kinase inhibitors are considered as potential drugs in inflammatory diseases. Therefore, the effect of endotoxin administration on the response of soluble(s) tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), a death receptor ligand, and the role of p38 MAP kinase inhibition was studied in 21 human volunteers. The volunteers received 30 min before the endotoxin infusion a single oral dose of placebo or the selective p38 MAP kinase inhibitor drug, RWJ-67657. Plasma sTRAIL increased 10-fold to 6564 +/- 511 pg/mL after 2.5 h. This increase was blocked completely by the highest dose of RW-J6765.This is the first report showing that endotoxin increases sTRAIL where the p38 MAP kinase signaling pathway is involved.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of tumor cells but not normal cells; its role in normal nontransformed tissues is unknown. We report here that chronic blockade of TRAIL in mice exacerbated autoimmune arthritis, and that intraarticular TRAIL gene transfer ameliorated the disease. In vivo, TRAIL blockade led to profound hyperproliferation of synovial cells and arthritogenic lymphocytes and heightened the production of cytokines and autoantibodies. In vitro, TRAIL inhibited DNA synthesis and prevented cell cycle progression of lymphocytes. Interestingly, TRAIL had no effect on apoptosis of inflammatory cells either in vivo or in vitro. Thus, unlike other members of the tumor necrosis factor superfamily, TRAIL is a prototype inhibitor protein that inhibits autoimmune inflammation by blocking cell cycle progression.     

Critical contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to apoptosis of human CD4+ T cells in HIV-1-infected hu-PBL-NOD-SCID mice

Apoptosis is a key for CD4+ T cell destruction in HIV-1-infected patients. In this study, human peripheral blood lymphocyte (PBL)-transplanted nonobese diabetic (NOD)-severe combined immunodeficient (SCID) (hu-PBL-NOD-SCID) mice were used to examine in vivo apoptosis after HIV-1 infection. As the hu-PBL-NOD-SCID mouse model allowed us to see extensive infection with HIV-1 and to analyze apoptosis in human cells in combination with immunohistological methods, we were able to quantify the number of apoptotic cells with HIV-1 infection. As demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), massive apoptosis was predominantly observed in virus-uninfected CD4+ T cells in the spleens of HIV-1-infected mice. A combination of TUNEL and immunostaining for death-inducing tumor necrosis factor (TNF) family molecules indicated that the apoptotic cells were frequently found in conjugation with TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD3+CD4+ human T cells. Administration of a neutralizing anti-TRAIL mAb in HIV-1-infected mice markedly inhibited the development of CD4+ T cell apoptosis. These results suggest that a large number of HIV-1-uninfected CD4+ T cells undergo TRAIL-mediated apoptosis in HIV-infected lymphoid organs.     

Down-regulation of protein kinase Ceta potentiates the cytotoxic effects of exogenous tumor necrosis factor-related apoptosis-inducing ligand in PC-3 prostate cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a highly promising candidate for the treatment of cancer because it elicits cell death in the majority of tumor cells while sparing most normal cells. Some cancers, however, display resistance to TRAIL, suggesting that treatment with TRAIL alone may be insufficient for cancer therapy. In the present study, we explored whether the apoptotic responsiveness of PC-3 prostate cancer cells to TRAIL could be enhanced by targeting the novel protein kinase C (PKC) isoform eta. Transfection of PC-3 cells with second-generation chimeric antisense oligonucleotides against PKCeta caused a time- and dose-dependent knockdown of PKCeta, as revealed by real-time RT-PCR and Western blot analyses. Knockdown of PKCeta resulted in a marked amplification of TRAIL's cytotoxic activity. Cell killing could be substantially prevented by the pan-caspase inhibitor z-VAD-fmk. In addition, PKCeta knockdown and administration of TRAIL significantly synergized in activation of caspase-3 and internucleosomal DNA fragmentation. Knockdown of PKCeta augmented TRAIL-induced dissipation of the mitochondrial transmembrane potential and release of cytochrome c from mitochondria into the cytosol, indicating that PKCeta acts upstream of mitochondria. We conclude that PKCeta represents a considerable resistance factor with respect to TRAIL and a promising target to exploit the therapeutic potential of TRAIL.     

急性排斥期大鼠角膜移植物肿瘤坏死因子相关凋亡诱导配体及死亡受体4表达变化与环孢素A的干预

目的:研究提示,肿瘤坏死因子相关凋亡诱导配体及其死亡受体4可能参与角膜移植免疫排斥反应.观察免疫抑制剂环孢素A对急性排斥期角膜移植物肿瘤坏死因子相关凋亡诱导配体及其受体死亡受体4表达的影响.方法:实验于2007-07/10在南方医科大学珠江医院中心实验室完成,对动物处置方法符合动物伦理学要求.选用Wistar大鼠18只为供体,SD大鼠36只为受体,钻取供体双眼角膜植片,于受体右眼行穿透性角膜移植术;另取5只Wistar大鼠做正常对照.按随机数字表法将受体大鼠分为2组(n=18),即生理盐水组及环孢素A组,术后药物滴眼,2次/d,1滴/次,连用2周.应用免疫组织化学法检测急性排斥期角膜植片肿瘤坏死因子相关凋亡诱导配体及死亡受体4表达情况.结果:36只受体大鼠全部进入结果分析.①肿瘤坏死因子相关凋亡诱导配体及死亡受体4在正常角膜均有表达.主要分布于上皮层,在基质层及内皮层有少量表达.②环孢素A组和生理盐水组大鼠角膜植片各层肿瘤坏死因子相关凋亡诱导配体及死亡受体4表达均增高,生理盐水组增高尤为明显.结论:肿瘤坏死因子相关凋亡诱导配体及死亡受体4的表达参与角膜移植免疫排斥反应的发生,环孢素A可通过下调其表达抑制角膜移植免疫排斥反应.     

Late expression of p53 from a replicating adenovirus improves tumor cell killing and is more tumor cell specific than expression of the adenoviral death protein

Gene transfer of p53 induces cell death in most cancer cells, and replication-defective adenoviral vectors expressing p53 are being evaluated in clinical trials. However, low transduction efficiency limits the efficacy of replication-defective vector systems for cancer therapy. The use of replication-competent vectors for gene delivery may have several advantages, holding the potential to multiply and spread the therapeutic agent after infection of only a few cells. However, expression of a transgene may adversely affect viral replication. We have constructed a replicating adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle and also contains a deletion of the adenoviral death protein (ADP). Adp53rc-infected cancer cells demonstrated high levels of p53 expression in parallel with the late expression pattern of the adenoviral fiber protein. p53 expression late in the viral life cycle did not impair effective virus propagation. Survival of several lung cancer cell lines was significantly diminished after infection with Adp53rc, compared with an identical p53-negative control virus. p53 expression also improved virus release and spread. Interestingly, p53 was more cytotoxic than the ADP in cancer cells but less cytotoxic than the ADP in normal cells. In conclusion, late expression of p53 from a replicating virus improves tumor cell killing and viral spread without impairing viral replication. In addition, in combination with a deletion of the ADP, specificity of tumor cell killing is improved.     

Curcumin sensitizes prostate cancer cells to tumor necrosis factor-related apoptosis-inducing ligand/Apo2L by inhibiting nuclear factor-kappaB through suppression of IkappaBalpha phosphorylation

Epidemiologic studies suggest that diet rich in plant-derived foods plays an important role in the prevention of prostate cancer. Curcumin, the yellow pigment in the spice turmeric, has been shown to exhibit chemopreventive and growth inhibitory activities against multiple tumor cell lines. We have shown previously that curcumin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L interact to induce cytotoxicity in the LNCaP prostate cancer cell line. In this study, we investigated the mechanism by which curcumin augments TRAIL-induced cytotoxicity in LNCaP cells. Subtoxic concentrations of the curcumin-TRAIL combination induced strong apoptotic response in LNCaP cells as demonstrated by the binding of Annexin V-FITC and cleavage of procaspase-3. Furthermore, LNCaP cells express constitutively active nuclear factor-kappaB (NF-kappaB), which is inhibited by curcumin. Because NF-kappaB has been shown to mediate resistance to TRAIL-induced apoptosis in tumor cells, we investigated whether there is a relationship between NF-kappaB activation and resistance to TRAIL in LNCaP prostate cancer cells. Pretreatment with curcumin inhibited the activation of NF-kappaB and sensitized LNCaP cells to TRAIL. A similar increase in the sensitivity of LNCaP cells to TRAIL-induced apoptosis was observed following inhibition of NF-kappaB by dominant negative mutant IkappaBalpha, an inhibitor of NF-kappaB. Finally, curcumin was found to inhibit NF-kappaB by blocking phosphorylation of IkappaBalpha. We conclude that NF-kappaB mediates resistance of LNCaP cells to TRAIL and that curcumin enhances the sensitivity of these tumor cells to TRAIL by inhibiting NF-kappaB activation by blocking phosphorylation of IkappaBalpha and its degradation.     

Association of post-cardioversion transcardiac concentration gradient of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) and inflammatory biomarkers to atrial fibrillation recurrence

Objectives

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) has been shown to have both pro- and anti-apoptotic activities and is associated to better prognosis in heart failure. The aim of this study was to determine the transcardiac concentration gradient of sTRAIL and inflammatory biomarkers after AF cardioversion and assess their relation to AF recurrence.

Design and methods

We measured transcardiac gradients (coronary sinus concentration minus aortic root concentration) of sTRAIL, C-reactive protein (hsCRP) and interleukin-6 (IL-6) in patients with non-valvular AF after electrical cardioversion. Six-month AF recurrence was the study endpoint.

Results

There were no differences in sTRAIL and hsCRP concentrations in peripheral venous blood between patients with and without AF recurrence (p = 0.066 and 0.149, respectively), while IL-6 was higher in patients with recurrence (p = 0.032). Only sTRAIL showed a significant transcardiac gradient [3 pg/mL (IQR 1–4 pg/mL); p = 0.01]. sTRAIL gradient was 4 pg/mL (IQR 3–5 pg/mL) in patients without recurrence versus − 1 pg/mL (IQR − 2–1 pg/mL) in those with recurrence (p < 0.001). IL-6 (p = 0.281) and hsCRP (p = 0.979) aortic concentrations were not significantly different from coronary sinus concentrations. In multivariate analysis, sTRAIL transcardiac gradient (beta − 0.81, p = 0.004) remained a negative predictor of AF recurrence.

Conclusion

This study demonstrates the existence of a significant transcardiac sTRAIL concentration gradient in patients with non-valvular AF, inversely associated to AF recurrence. These results suggest production of sTRAIL by the heart and a protective role against substrate-altering processes in AF-prone atria. This could have implications for TRAIL-targeting therapies currently under development.     

Xylocydine, a novel inhibitor of cyclin-dependent kinases, prevents the tumor necrosis factor-related apoptosis-inducing ligand-induced apoptotic cell death of SK-HEP-1 cells

Xylocydine (4-amino-6-bromo-7-(beta-l-xylofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamide) blocks cyclin-dependent kinase CDK1 and CDK2/cyclin A activity in vitro (IC(50) 1.4 and 61 nM, respectively) while minimally inhibiting the three other Ser/Thr protein kinases tested (IC(50) 21-86 microM). Reduced phosphorylated nucleolin and retinoblastoma protein levels showed it also efficiently inhibited cellular CDK1 and CDK2 activity (IC(50) 50-100 and 200-500 nM, respectively). Moreover, it blocked the functional activity of CDKs in tumor necrosis factor-related apoptosis-inducing ligand-induced SK-HEP-1 cell apoptosis 20 to 1000-fold more potently than olomoucine and roscovitine. Xylocydine is thus a novel and potent CDK inhibitor that could be used to interfere with cell cycle- and apoptosis-related CDK activity in various diseases.     

原发性胆汁性肝硬化患者外周血TRAIL基因表达水平初探

目的建立检测人肿瘤坏死因子相关凋亡诱导配体（TRAIL）mRNA的实时荧光相对定量方法,探讨其基因表达与原发性胆汁性肝硬化（PBC）的相关性。方法基于TaqMan—MGB探针技术,以B—actin基因为内参照,通过目的基因与内参基因荧光强度达到一定阈值时的循环数之差,即△Ct值定量原始模板浓度,建立实时荧光逆转录聚合酶链反应（FQ—RT—PCR）相对定量方法,检测30例PBC患者、25例乙型肝炎（简称乙肝）后肝硬化患者及30名正常人外周血单个核细胞（PBMC）TRAIL基因的表达,同时检测血浆γ－谷氨酰转肽酶（GGT）和碱性磷酸酶（ALP）浓度,并作相关性分析：结果PBC患者PBMCTRAIL基因检测的△Ct值为2．4±1．2,其相对表达量是正常对照组的6．96倍,差异有统计学意义（P〈0．05）,乙肝后肝硬化患者TRAIL基因△Ct值为3．3±1．7,其相对表达量是正常对照组的3．73倍,差异有统计学意义（P〈0．05）,而PBC组与乙肝后肝硬化组比较,差异无统计学意义（P〉0．05）。PBC患者TRAIL的△Ct值与GGT浓度的对数值呈显著相关（r=－0．396,P〈0．05）。结论PBC患者及乙肝后肝硬化患者PBMC TRAIL基因表达水平明显升高,提示TRAIL在自身免疫性肝病及肝脏病毒损伤中均具有重要作用。     

原发性胆汁性肝硬化患者外周血TRAIL基因表达水平初探

目的建立检测人肿瘤坏死因子相关凋亡诱导配体(TRAIL)mRNA的实时荧光相对定量方法,探讨其基因表达与原发性胆汁性肝硬化(PBC)的相关性。方法基于TaqMan-MGB探针技术,以β-actin基因为内参照,通过目的基因与内参基因荧光强度达到一定阈值时的循环数之差,即△Ct值定量原始模板浓度,建立实时荧光逆转录聚合酶链反应(FQ-RT-PCR)相对定量方法,检测30例PBC患者、25例乙型肝炎(简称乙肝)后肝硬化患者及30名正常人外周血单个核细胞(PBMC)TRAIL基因的表达,同时检测血浆γ-谷氨酰转肽酶(GGT)和碱性磷酸酶(ALP)浓度,并作相关性分析。结果PBC患者PBMC TRAIL基因检测的△Ct值为2.4±1.2,其相对表达量是正常对照组的6.96倍,差异有统计学意义(P<0.05),乙肝后肝硬化患者TRAIL基因△Ct值为3.3±1.7,其相对表达量是正常对照组的3.73倍,差异有统计学意义(P<0.05),而PBC组与乙肝后肝硬化组比较,差异无统计学意义(P>0.05)。PBC患者TRAIL的△Ct值与GGT浓度的对数值呈显著相关(r=-0.396,P<0.05)。结论PBC患者及乙肝后肝...     

可溶性肿瘤坏死因子相关凋亡诱导配体在原发性胆汁性肝硬化患者血清中的表达及意义

目的观察人可溶性肿瘤坏死因子相关凋亡诱导配体（sTRAIL）在原发性胆汁性肝硬化（PBC）患者外周血中水平的表达,并探讨sTRAIL在PBC发病机制中的作用。方法用酶联免疫吸附测定（ELISA）法检测PBC患者26例（PBC组）,慢性肝炎肝硬化患者27例（慢性肝炎肝硬化组）以及健康体检者25例（正常对照组）的sTRAIL并同时测定IgG、IgA、IgM,观察各指标在PBC中的改变。结果PBC组和慢性肝炎肝硬化组sTRAIL浓度均显著高于正常对照组（158．73±42．45）pmol／L,（108．13±41.60）pmol／L vs（73．83±8．60）pmol／L（P〈0．01）。PBC组sTRAIL也较慢性肝炎肝硬化组明显升高（P〈0．01）。结论sTRAIL在PBC患者及慢性肝炎肝硬化患者血清中均升高,但二者升高程度比较差异有统计学意义,检测sTRAIL对PBC患者的,临床诊断中具有辅助作用并有助于PBC发病机制的研究。     

Dynamics of tumor cell killing by human T lymphocytes armed with an anti-carcinoembryonic antigen chimeric immunoglobulin T-cell receptor

Chimeric immunoglobulin T-cell receptors (IgTCR) join the antigen-binding portion of an antibody to one of the signaling chains of the TCR. A previous report described the construction and functional testing of an IgTCR gene directed against the carcinoembryonic tumor antigen (CEA). These preclinical studies showed the proper assembly and cell surface expression of anti-CEA IgTCR molecules, specific target antigen binding, and activation of T-cell effector functions. Although IgTCR-modified T cells function well in vitro, therapeutic applications in humans may be complicated by various factors, such as the availability of appropriate T-cell cytokines, high systemic levels of antagonistic soluble CEA, and antigenic diversity in tumor cell populations. The current study analyzes tumor cell killing by IgTCR-modified human T cells under conditions that more closely model those that may be encountered in persons with cancer. This analysis shows that 1) depriving IgTCR-modified T cells of interleukin-2 does not diminish anti-CEA cytotoxic T lymphocyte activity, but does eliminate killing by lymphokine-activated killer cells; 2) high levels of soluble CEA do not significantly inhibit tumor cell killing even when approximately 80% of the chimeric receptors are blocked; and 3) CEA+ tumor cells that can down-regulate cell surface CEA evade immune destruction by IgTCR-modified T cells. These results have important implications for application strategies and protocol design considerations for early clinical testing of IgTCR anti-tumor therapies.     

Camptothecin- and etoposide-induced apoptosis in human leukemia cells is independent of cell death receptor-3 and -4 aggregation but accelerates tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death

During camptothecin- and etoposide (VP-16)-induced apoptosis in HL-60 cells, the expression level of cell death receptor-3 (DR3), cell death receptor-4 (DR4), and FAS remained mostly unchanged, whereas the expression of silencers of death domain (SODD) and FLICE inhibitory proteins, inhibitors of the cell death receptor signaling pathways, decreased substantially. By indirect immunofluorescence and immunoperoxidase imaging and with gel filtration column chromatography, we observed rapid aggregation at the cell surface and the appearance of high molecular weight protein complexes primarily involving DR3, and DR3 and DR4 after camptothecin and VP-16 treatment, respectively. Both drugs failed to rapidly promote FAS aggregation in these cells. The high expression level of SODD or of dominant negative forms of FADD (FADD-DN) and DAP3 (DAP3-DN), or of NH2-terminal deletion mutant of TRADD (TRADD-ND) achieved by transient transfection experiments, did not impair the kinetics of apoptosis after camptothecin and VP-16 treatment in HL-60 and U937 cells. Taken together, these observations suggested that camptothecin and VP-16 induced rapid aggregation of DR4 and DR3, but paradoxically, the importance of these events in signaling apoptosis is uncertain, because the kinetics of apoptosis were unaffected, even in the presence of a high expression level of SODD, FADD-DN, TRADD-ND, and DAP3-DN. However, camptothecin or VP-16 treatment in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) substantially accelerated kinetics of apoptosis than treatment with camptothecin, VP-16, or TRAIL alone. In contrast, cotreatment of camptothecin or VP-16 with TWEAK or TL1A did not facilitate apoptosis in HL60 cells. These findings suggest that DR4 aggregation mediated by camptothecin or VP-16 could represent a mean that accelerates TRAIL-induced apoptosis.     

Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety

Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor gene family known to induce apoptosis in a number of cancer cell lines and may have broad-spectrum activity against human malignancies. These studies have evaluated the potency of recombinant soluble human Apo2L/TRAIL in a mouse xenograft model and the disposition and safety of Apo2L/TRAIL in rodents and nonhuman primates. Mice with established COLO205 tumors were given daily i.v. injections of Apo2L/TRAIL (30-120 mg/kg/day). Control tumors doubled in size every 2 to 3 days, while time to tumor doubling in the treatment groups was significantly longer and related to dose (14-21 days). For pharmacokinetic studies, Apo2L/TRAIL was given as an i.v. bolus to mice (10 mg/kg), rats (10 mg/kg), cynomolgus monkeys (1, 5, and 50 mg/kg), and chimpanzees (1 and 5 mg/kg). Apo2L/TRAIL was rapidly eliminated from the serum of all species studied. Half-lives were approximately 3 to 5 min in rodents and approximately 23 to 31 min in nonhuman primates. Allometric scaling provided estimates of Apo2L/TRAIL kinetics in humans, suggesting that on a milligram per kilogram basis, doses significantly lower than those used in xenograft studies could be effective in humans. Apo2L/TRAIL clearance was highly correlated with glomerular filtration rate across species, indicating that the kidneys play a critical role in the elimination of this molecule. Safety evaluations in cynomolgus monkeys and chimpanzees revealed no abnormalities associated with Apo2L/TRAIL exposure. In conclusion, these studies have characterized the disposition of Apo2L/TRAIL in rodents and primates and provide information that will be used to predict the pharmacokinetics of Apo2L/TRAIL in humans.