Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells

Proteinase-activated receptors (PARs) can activate a number of signalling events, including T-cell signal-transduction pathways. Recent data suggest that the activation of PARs 1, 2 and 3 in Jurkat T-leukaemic cells induces tyrosine phosphorylation of the haematopoietic signal transducer protein, VAV1. To activate the PARs, this study used the agonist peptides SFLLRNPNDK, SLIGKVDGTS and TFRGAPPNSF, which are based on the sequences of the tethered ligand sequences of human PARs 1, 2 and 3, respectively. Here, we show that peptides based on either the human or murine PAR(3)-derived tethered ligand sequences (TFRGAP-NH(2) or SFNGGP-NH(2)) do not activate PAR(3), but rather activate PARs 1 and 2, either in Jurkat or in other PAR-expressing cells. Furthermore, whilst thrombin activates only Jurkat PAR(1), trypsin activates both PARs 1 and 2 and also disarms Jurkat PAR(1) for thrombin activation. We conclude therefore that in Jurkat or related T cells, signalling via PARs that can affect VAV1 phosphorylation is mediated via PAR 1 or 2, or both, and that distinct serine proteinases may potentially differentially affect T-cell function in the settings of inflammation.     

Low-dose doxorubicin-induced necrosis in Jurkat cells and its acceleration and conversion to apoptosis by antioxidants

We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2-arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine beta-d-arabinofuranoside for 60 h induced necrosis both in Jurkat and ml-1 cells. Pre-necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre-apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin-induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n-acetyl-l-cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis-resistant Jurkat subclone was also refractory to doxorubicin-induced necrosis, apoptosis and necrosis might share some common pathways. Low-dose doxorubicin increased micronuclei-positive cell percentages and also suppressed high-dose doxorubicin-induced apoptosis in Jurkat and ml-1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.     

蝎素组分Ⅲ对Jurkat E6-1细胞中LAT和SLP-76表达的影响

目的研究蝎素组分Ⅲ(SVC-Ⅲ)对Jurkat E6-1细胞LAT和SLP-76表达的影响,并探讨SVC-Ⅲ参与细胞免疫调控的可能机制。方法不同浓度SVC-Ⅲ(100 ng.mL-1、10 ng.mL-1、1 ng.mL-1、0.1 ng.mL-1)与植物凝集素(PHA)共同刺激培养Jurkat E6-1细胞。72 h后收获细胞,利用逆转录聚合酶链反应(RT-PCR)技术检测细胞内信号分子LAT和SLP-76 mRNA表达变化情况。结果高浓度SVC-Ⅲ(100 ng.mL-1)明显抑制Jurkat E6-1细胞LAT和SLP-76的mRNA的表达(P<0.05),低浓度SVC-Ⅲ(0.1 ng.mL-1、1 ng.mL-1、10 ng.mL-1)对Jurkat E6-1细胞LAT和SLP-76的mRNA的表达没有明显作用(P>0.05)。结论SVC-Ⅲ能够抑制Jurkat E6-1细胞LAT和SLP-76的表达,在一定程度上阻止T细胞信号进一步的传递,从而抑制Jurkat E6-1细胞的活化能力。并且SVC-Ⅲ可抑制PHA诱导的Jurkat E6-1细胞的活化,其作用机制可能与早期活化相关的LAT和SLP-76等的表达相关。     

Ranking the toxicity of fatty acids on Jurkat and Raji cells by flow cytometric analysis

The fatty acids have an important role in the control of leukocyte metabolism and function. Higher concentrations of certain fatty acids, particularly polyunsaturated fatty acids (PUFAs) and volatile fatty acids, can cause cell death via apoptosis or, when concentrations are greater, necrosis. In this study, we determined the highest concentrations of various fatty acids that are non-toxic to two human leukemic cell lines, Jurkat (T-lymphocyte) and Raji (B-lymphocyte). Toxicity was evaluated by either loss of membrane integrity and/or DNA fragmentation using flow cytometric analysis. There were no remarkable differences for the toxicity of the fatty acids between B and T cell lines. The cytotoxicity of the fatty acids was related to the carbon chain length and number of double bonds: docosahexaenoic ACID=eicosapentaenoic ACID=arachidonic ACID=γ-linolenic ACID=stearic ACID=palmitic acid > linoleic ACID=palmitoleic acid > vacenic ACID=lauric acid > oleic acid > elaidic acid > capric acid > butyric acid > caprylic ACID=caproic ACID=propionic acid. The proportion of cells undergoing apoptosis or necrosis, induced by the fatty acids tested, remains to be investigated.     

HIV-1慢性感染Jurkat细胞系的建立及其特性研究

目的建立艾滋病病毒Ⅰ型（HIV-1）慢性感染Jurkat细胞系,研究其生物学特性。方法参照文献方法改良,建立HIV-1ⅢB慢性感染Jurkat细胞系。其细胞系特性采用以下方法检测：光学显微镜观察细胞形态;流式细胞仪检测HIV-1ⅢB感染细胞的阳性率;噻唑蓝（MTT）法检测抗病毒药物对慢性感染细胞的毒性作用;酶联免疫吸附试验（ELISA）检测药物对慢性感染细胞中病毒复制的影响;合胞体形成方法检测药物对HIV-1ⅢB慢性感染细胞与正常细胞融合的阻断作用。结果成功建立了HIV-1慢性感染Jurkat细胞系（Jurkat/HIV-1ⅢB）,感染细胞阳性率〉90%。检测影响病毒复制各周期的抗病毒药物对Jurkat/HIV-1ⅢB细胞的作用,发现Jurkat/HIV-1ⅢB细胞具有HIV-1慢性感染细胞的生物学特征。结论成功建立Jurkat/HIV-1ⅢB细胞系,为抗HIV-1药物的研发和病毒感染机制研究提供了新的工具。     

二氟甲基鸟氨酸对T淋巴瘤Jurkat细胞生长的影响

目的研究二氟甲基鸟氨酸(difluromethylornithine,DFMO)对人T淋巴瘤Jurkat细胞的影响及其作用机制,为探讨DFMO能否用于治疗人白血病提供实验依据。方法DFMO(0～10mmol/L)处理人T淋巴瘤Jurkat细胞24～72h后,MTS法检测细胞的存活率,化学分析法检测精胺氧化酶(spermine oxidase,SMO)和乙酰多胺氧化酶(acetylpolyamine oxidase,APAO)活性,DNA片段化分析检测细胞凋亡,荧光染料法检测细胞线粒体膜电位变化,Western blot法检测细胞内Bax含量,分光光度法检测caspase-3酶活性。结果DFMO处理细胞能显著抑制Jurkat细胞的生长(P<0.01),抑制率随药物浓度的增加和作用时间的延长而加大,DFMO浓度为10mmol/L时,24h抑制率为33%,48h抑制率为38%,72h抑制率为49%。DFMO处理可导致Jurkat细胞DNA片段化,促进Bax由细胞质向线粒体转移,细胞内caspase-3活性增加(增加46%,P<0.05),并伴有线粒体膜电位的显著降低,DFMO浓液为2和5mmol/L时,分别降低29...     

七叶皂苷钠抑制人白血病Jurkat细胞增殖的机制研究

目的 研究七叶皂苷钠抑制人白血病Jurkat细胞增殖的作用及其机制。方法 MTT法分析七叶皂苷钠对Jurkat细胞增殖的抑制作用,Hoechst 33258染色、FITC-Annexin V/PI双染、DNA Ladder、流式细胞术检测细胞凋亡和细胞周期,Western blotting法分析凋亡相关蛋白变化。结果 七叶皂苷钠呈质量浓度和时间相关方式抑制Jurkat细胞增殖;经七叶皂苷钠处理后的Jurkat细胞出现凋亡的形态学特征、DNA条带,Annexin V⁺/PI^?细胞（早期凋亡细胞）显著增加;七叶皂苷钠可活化Jurkat细胞中Caspase-8、Caspase-9、Caspase-3,引起PARP的切割,并减少Bcl-2蛋白的表达。结论 七叶皂苷钠能有效地通过诱导细胞凋亡抑制Jurkat细胞增殖。     

The involvement of ROS overproduction and mitochondrial dysfunction in PBDE-47-induced apoptosis on Jurkat cells

2,2′,4,4′-Tetrabromodiphenyl ether (PBDE-47), as one of the congeners of polybrominated diphenyl ethers (PBDEs), is widely present and threatens the human health in many aspects. This study aims to investigate the toxic effects of PBDE-47 on cell viability, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of Jurkat cells in vitro. The results showed that PBDE-47 significantly inhibited the viability of Jurkat cells in a dose-dependent manner by alamar blue assay. Significant induction of apoptosis was detected in Jurkat cells at 25-100 μM by propidium iodide staining, accompanied with overproduction of ROS and downregulation of MMP. Furthermore, N-acetyl-L-cysteine (NAC), a widely used ROS scavenger, significantly reduced the PBDE-47-induced apoptosis by decreasing ROS level and mediating recovery of the MMP. In conclusion, the results of this study suggest that PBDE-47 could induce apoptosis in Jurkat cells and ROS and mitochondrial dysfunction play important roles in the apoptotic process.