Application of flow cytometry to molecular medicine: detection of tumor necrosis factor-related apoptosis-inducing ligand receptors in acute myeloid leukaemia blasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF (tumor necrosis factor) family, is currently regarded as a potential anti-cancer agent. Nevertheless, several types of cancer cells display a low sensitivity to TRAIL or are completely resistant to this pro-apoptotic cytokine. TRAIL signalling is dependent on four receptors. Two of them, death receptors 4 and 5 (DR4 and DR5), induce apoptosis, whereas decoy receptors 1 and 2 (DcR1 and DcR2) are unable to evoke cell death upon TRAIL binding. TRAIL resistance may be related to the expression of TRAIL decoy receptors. TRAIL has been proposed as a novel therapeutic agent for the treatment of haematological disorders, including acute myeloid leukaemia (AML). Surprisingly, however, very limited information is available concerning the expression of TRAIL receptors in AML blasts. Here, we have evaluated, using flow cytometry, TRAIL receptor surface expression and sensitivity to TRAIL-dependent apoptosis of AML blasts from 30 patients. We observed frequent expression of TRAIL DcR1 and DcR2, while expression of DR4 and DR5 was less frequent. Nevertheless, the expression of DR4 or DR5 in leukaemic cells was always matched by a similar expression of one of the decoy receptors. Leukaemic blasts were invariably resistant, even to a high concentration (1000 ng/ml) of TRAIL. We suggest that AML blasts are resistant to TRAIL apoptosis in vitro. Therefore, it is unlikely that TRAIL alone might be used in the future as an innovative pharmacological agent for the treatment of AML.     

Characterization of tumour necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in the adult human testis

Tumour necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumour necrosis factor-alpha (TNF-alpha) family of cytokines which is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated by immunohistochemistry in adult human testes. In addition, TRAIL and its receptors were studied in terms of protein and mRNA using western blot analysis and RT-PCR respectively. TRAIL and its receptors were immunodetected according to the different testicular cell types: TRAIL, DR5/TRAIL-R2 and DcR2/TRAIL-R4 were localized in Leydig cells, DR4/TRAIL-R1 was seen in peritubular and Sertoli cells whereas ligand and all receptors were detected in germ cells. Proteins and mRNA corresponding to TRAIL and its receptors were also identified in adult human testes. In conclusion, TRAIL and its receptors DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1/TRAIL-R3 and DcR2/TRAIL-R4 are expressed in the human testis, and are predominantly localized in different germ cell types.     

Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa,adenomas, and carcinomas

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cell lines. Four membrane-bound receptors for TRAIL have been identified, two apoptosis-mediating receptors, DR4 and DR5, and two apoptosis-inhibiting receptors, DcR1 and DcR2. The aim of this study was to examine the role of TRAIL and its receptors in colorectal cancer development. The immunohistochemical expression and localization of TRAIL and its receptors were investigated in normal mucosa (n=10), adenomas (n=19), and carcinomas (n=21). Correlations between the expression of TRAIL and its receptors and the degree of apoptosis (assessed by M30 expression) and histopathological characteristics were explored. TRAIL and its receptors were expressed in normal mucosal epithelium. Expression of the receptors was seen in adenomas and carcinomas. TRAIL expression was lost in a subset of colorectal tumours, more frequently in carcinomas than in adenomas (p<0.05). DR4 and DR5 staining was stronger in neoplastic cells than in normal cells and was accompanied by a higher degree of apoptosis. No differences were found between tumour and normal cells regarding DcR1 and DcR2 expression. No correlations were found between TRAIL or TRAIL receptor expression and histopathological characteristics. In conclusion, marked changes were seen in the course of the adenoma-carcinoma sequence with respect to the expression of TRAIL and TRAIL receptors DR4 and DR5. The stronger expression of DR4 and DR5 in neoplastic cells than in normal cells, together with a higher degree of apoptosis, suggests a possible functional role for these receptors in apoptosis induction in neoplastic colorectal cells.     

Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppression of caspases-8, 7, and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the latest members of the TNF superfamily known to induce apoptosis in a wide variety of tumor cells. Some cell types, however, are quite resistant to TRAIL. We investigated the effect of ectopic expression of Bcl-2 and Bcl-xL on TRAIL-induced apoptosis in human acute myelogenous leukemia HL-60 cells. We found that HL-60 cells, which express TRAIL receptors (also called death receptor, DR) DR4, DR5, and Dc (decoy) R2, are highly sensitive to TRAIL-induced cytotoxicity. Greater than 90% killing occurred within 24 h of TRAIL treatment. The expression of Bcl-2 and Bcl-xL, however, completely abolished the TRAIL-induced cytotoxic effects. Treatment of HL-60 cells with TRAIL induced caspase-8 activation within 2-4 h, but no activation could be seen in Bcl-2-expressing or Bcl-xL-expressing cells. TRAIL also induced cleavage of BID, which was also abolished by Bcl-2 and Bcl-xL. Similarly, TRAIL activated caspase-3 and caspase-7 in control cells but not in cells expressing Bcl-2 or Bcl-xL. Cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), was abrogated by ectopic expression of Bcl-2 and Bcl-xL. Inhibition of caspases by the pan-caspase inhibitor, benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone (zVAD-fmk) abolished the TRAIL-induced apoptosis. Overall, these results indicate that TRAIL-induced apoptosis involves activation of caspase-8, caspase-7, caspase-3, and BID cleavage, and Bcl-2 and Bcl-xL prevents TRAIL-induced apoptosis by abrogating caspase activation and BID cleavage.     

重组人可溶性TRAIL分子诱导白血病细胞株凋亡的研究

比较重组人可溶性TRAIL(rhsTRAIL)诱导Jurkat细胞株、K562细胞株以及HL-60细胞株凋亡之间的差异,探讨这些差异与细胞表面TRAIL受体(DR4、DR5、DcR1和DcR2)表达量的关系。不同浓度的rhsTRAIL分别处理Jurkat细胞、K562细胞和HL-60细胞12 h、24 h和48 h后,用流式细胞仪检测经碘化丙啶(PI)染色后的细胞凋亡情况;用RT-PCR方法检测细胞表面受体DR4、DR5、DcR1、DcR2的表达。培养12 h、24 h、48 h后,不同浓度rhsTRAIL诱导Jurkat细胞株的凋亡率均明显高于对照组,且具有剂量依赖性和时间依赖性;但K562细胞株和HL-60细胞株未见明显的凋亡发生。RT-PCR结果显示,培养12 h、24 h、48 h后,Jurkat细胞株表面DR4的表达随时间的延长和rhsTRAIL浓度的升高而升高,而DR5、DcR1和DcR2的表达未检出;K562和HL-60细胞株表面DR4的表达没有明显变化,而且DR5、DcR1和DcR2的表达也未检出。rhsTRAIL诱导Jurkat细胞株的凋亡具有剂量依赖性和时间依赖性,且与其细胞表面DR4的表达呈正相关;在一定的浓度条件下,rhsTRAIL未能诱导K 562和HL-60细胞株发生明显凋亡,且其细胞表面DR4的表达也未见明显变化。这些结果提示,应用TRAIL治疗不同种类白血病时,应注意它的使用剂量和适应范围。     

Progression in melanoma is associated with decreased expression of death receptors for tumor necrosis factor-related apoptosis-inducing ligand

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in melanoma by interaction with death receptors TRAIL-R1 (DR4) or TRAIL-R2 (DR5) on melanoma cells or resists apoptosis by interaction with decoy receptors TRAIL-R3 (DcR1) or TRAIL-R4 (DcR2). Studies on cell lines suggest that there is a wide variation in TRAIL death receptor expression; however, their expression on excised human melanoma is not well documented. In view of this, we studied death receptor expression on melanomas using monoclonal antibodies specific for these receptors. Immunohistochemical staining for DR4, DR5, and DcR1/DcR2 was performed on formalin-fixed paraffin-embedded sections of 100 cases of primary melanoma, metastatic melanoma, and benign nevi. Percentage expressions of DR4 versus DR5 in benign nevi, primary melanoma, and melanoma metastases were 40% versus 90%, 69% versus 98%, and 55% versus 66%, respectively. There were significant differences in the mean percentage of DR5-positive cells between different groups of melanocytic lesions. Percent expression was higher in thin (< or =1.0 mm) compared with thick primary melanoma (88.9% versus 66.9%), and expression was less in subcutaneous metastases (49%) and lymph node metastases (30.6%) (P < .005). Expression was also higher in compound nevi (57%) than dysplastic nevi (49%). DcR1/DcR2 was found in 75% of benign nevi, 62% of primary melanomas, and 74% melanoma metastases. The results showed a wide variation in the expression of death receptors for TRAIL between and within primary and metastatic melanoma and a decreased expression on the thick primary melanoma and metastatic melanoma. This suggests that melanoma may not respond to treatment with TRAIL unless given with agents that increase the expression of TRAIL death receptors.     

TRAIL受体在肿瘤细胞系上的表达及意义

目的 检测TNF相关凋亡诱导配体（TRAIL）的受体，在来源于血液系统、肝脏、肺脏和大肠的8个肿瘤细胞系中的表达，并探讨其意义。方法 采用半定量RT－PCR，对TRAIL受体的表达进行半定量检测。结果 TRAIL凋亡通路中，能够诱导凋亡反应的死亡受体DR4和DR5，在所检测的肿瘤细胞系中都有表达，其中DR5在所有肿瘤细胞系中的表达水平均显著高于DR4（P＜0．05）。而能够竞争性与TRAIL诱导的凋亡反应的诱骗受体DcR1和DcR2，在所有的肿瘤细胞中都呈低水平表达或不表达。结论 DR5可能在TRAIL诱导凋亡的通路中发挥最重要的作用。TRAIL死亡受体和诱骗受体在肿瘤细胞系中的表达具有差异性，这种差异性可在一定程度上解释不同细胞对TRAIL诱导凋亡的敏感度。     

慢性乙肝患者外周血单个核细胞TRAIL受体表达及其临床意义

目的探索慢性乙肝患者外周血单个核细胞(PBMC)肿瘤细胞坏死因子相关的凋亡诱导配体(TRAIL)受体在PBMC凋亡中的作用,及其与机体肝脏损伤的相关性。方法用RT-PCR和流式细胞术检测55例慢性乙肝患者(包括轻度、中度、重度)外周血单个核细胞TRAIL各受体(DR4、DR5、DcR1和DcR2)表达水平,同时检测肝功能相关指标,并进行相关性分析。以30例正常人作为对照。结果诱骗受体DcR1在慢性乙肝患者中的表达显著低于对照组(P<0.05)。DcR1的表达随慢性乙肝病情的加重而逐渐降低。慢性乙肝患者的DcR1表达与转氨酶(ALT)呈显著负相关,与血清白蛋白成显著正相关。结论慢性乙肝患者外周血单个核细胞DcR1表达下调可能是其细胞凋亡增加的机制之一,且DcR1表达情况可从一定程度上反映肝脏的损伤程度。     

Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-beta1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.     

TRAIL及其受体DR5与动脉粥样硬化关系的初步研究

目的探讨肿瘤坏死因子(INF)相关的凋亡诱导配体(TRAIL)及其受体DR5与动脉粥样硬化(AS)之间的关系。方法冠心病(CAD)组61例,正常对照组22例。应用酶联免疫吸附法测定血浆可溶性TRAIL(sTRAIL)和可溶性DR5(sDR5)水平。免疫组织化学测定冠状动脉TRAIL和DR5蛋白表达情况。结果CAD组血浆sTRAIL和sDR5水平均显著性升高(P<0.001,P<0.05)。3支血管病变组和双支血管病变组血浆sTRAIL和sDR5水平均分别显著高于单支血管病变组和正常对照组(P<0.01,P<0.01,P<0.01,P<0.05)。TRAIL和DR5主要表达于平滑肌细胞的胞质,TRAIL还可表达于AS中的巨噬细胞。AS组冠状动脉的TRAIL和DR5表达明显高于非AS组(P<0.01,P<0.05)。结论TRAIL及其受体DR5可能参与了AS的进展,其浓度越高,冠状动脉病变越重。     

Normal human primary CD4+ T lymphocytes synthesize and release functional osteoprotegerin in vitro

Osteoprotegerin (OPG) acts as a decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL). OPG regulates bone remodeling and the immune response. The primary objective was to decipher, among human peripheral blood mononuclear leukocytes (PBML) that produce OPG, the subset(s) responsible for this synthesis and its regulation. To this end, normal human PBML and CD4-, 8-, 19-, 14-enriched subpopulations were studied in vitro for OPG synthesis. PBML were subjected to adherence and immunomagnetic separation, and OPG expression was analyzed by PCR, northern and western blotting, and ELISA. The antiapoptotic effects of OPG were studied on TRAIL-stimulated RPMI 8226 myeloma cells. OPG was time-dependently produced by primary CD4+ T lymphocytes exclusively. OPG secretion was upregulated by anti-CD3 antibody stimulation or incubation with interleukin (IL)-4, IL-1beta, TNF-alpha, GM-CSF, and vitamin D(3). In contrast, IL-10 inhibited the basal and IL-4-induced production of OPG by T cells. Conditioned media from activated T lymphocytes decreased TRAIL-induced apoptosis of RPMI 8226 cells. This effect was reversed by addition of RANKL to the T-cell conditioned media. As human immunodeficiency virus-1 (HIV-1) targets CD4+ T cells, we evaluated the effects of recombinant HIV-1 gp120 proteins on OPG synthesis. The gp120 from three different HIV-1 strains significantly reduced the basal output of OPG from T cells. Furthermore, all four protease inhibitors (PIs) used in highly active antiretroviral therapy decreased OPG synthesis by human blood T cells, nelfinavir being the most efficient PI. The simultaneous presence of an HIV-1 gp120 and a PI abrogated the basal output of OPG. In conclusion, these results highlight a new role for T lymphocytes involved in pathologies. Activated CD4+ T cells could, through OPG release, have a paracrine effect on adjacent cells and contribute to reduce the local process of bone remodeling and cellular apoptosis.     

Trauma patients' elevated tumor necrosis related apoptosis inducing ligand (TRAIL) contributes to increased T cell apoptosis

Immunosuppression resulting from excessive post-trauma apoptosis of hyperactivated T cells is controversial. TRAIL mediated T cell apoptosis decreases highly activated T cells' responses. Caspase-10, a particular TRAIL target, was increased in trauma patients' T cells with concomitantly elevated plasma TRAIL levels. These patients' T cells developed anergy, implicating increased TRAIL-mediated T cell apoptosis in post-trauma T cell anergy. Control T cells cultured with patients' sera containing high TRAIL levels increased their caspase-10 activity and apoptosis. Stimulated primary T cells are TRAIL apoptosis resistant. Increased plasma thrombospondin-1 and T cell expression of CD47, a thrombospondin-1 receptor, preceded patients' T cell anergy. CD47 triggering of T cells increased their sensitivity to TRAIL-induced apoptosis. Augmentation of T cell TRAIL-induced apoptosis was secondary to CD47 triggered activation of the Src homology-containing phosphatase-1 (SHP-1) and was partially blocked by a SHP-1 inhibitor. We suggest that combined post-trauma CD47 triggering, SHP-1 mediated NFκB suppression, and elevated TRAIL levels increase patients' CD47 expressing T cell apoptosis, thus contributing to subsequent T cell anergy.     

DcR1 expression in endometrial carcinomas

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which mediates apoptosis by the extrinsic pathway. Up-regulation of decoy receptors, DcR1 and DcR2, may result in diminished binding of TRAIL to their functional receptors. DcR1 expression was assessed in normal endometrial tissue (NE) and endometrial carcinoma (EC) samples by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (PCR). IHC was performed in two tissue microarrays; one composed of 80 samples of NE and a second one constructed from paraffin-embedded blocks of 62 EC. For quantitative real-time RT-PCR analysis, RNA was obtained from 19 NE and 28 EC samples using Trizol®. mRNA expression of DcR1 was assessed with Taqman®-based assays in an Abi-Prism 700 SDS. Results were correlated with stage, histological type, and grade. By IHC, cytoplasmic expression of DcR1 was frequently seen in NE (79.6%) and varied according to the menstrual cycle. Positive DcR1 immunostaining was also detected in EC (98.1% of the cases) without any specific statistical association with histological type, grade, and stage. By quantitative real-time PCR, all NE had similar levels of DcR1expression (0.8–1.7 RQ), which were considered the basal levels of DcR1 expression in NE. Increased DcR1 expression (≥5-fold higher than the basal levels) was detected in 13 of 28 EC (46.4%). High DcR1 expression levels were found in ECs of different stages: IA, four of 12 (33%); IB, two of four (50%); IC, four of six (66%); and IIA and IIB three of six (50%). Results suggest that DcR1 expression occurs in a subset of EC and may contribute to resistance to TRAIL-induced apoptosis.     

IL-18 Upregulates the Production of Key Regulators of Osteoclastogenesis from Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

Recent data have demonstrated the importance of IL-18 in the induction and perpetuation of chronic inflammation in experimental arthritis. The aim of the present study was to elucidate whether IL-18 has any indirect effects on osteoclastogenesis by regulating the production of molecules from fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). Human FLS were isolated from RA synovial tissue and cultured in vitro for three to five passages. The expression of IL-18 receptor was determined by RT-PCR. The levels of soluble receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in culture supernatants were determined by ELISA. Membrane-bound RANKL expression was analyzed by flow cytometry. Both α and β chains of IL-18 receptor were confirmed in cultured FLS. IL-18 upregulated membrane-bound RANKL expression and soluble RANKL production by FLS in both time- and dose-dependent manners. In addition, IL-18 enhanced production of M-CSF, GM-CSF, and OPG from cultured FLS in a dose-dependent manner. IL-18 also increased the ratio of RANKL/OPG, suggesting that the net effect of IL-18 on FLS favors for the induction of osteoclast formation and bone resorption. In conclusion, IL-18 upregulates the production of key regulators of osteoclastogenesis from FLS in RA.