Mollugin induces apoptosis in human Jurkat T cells through endoplasmic reticulum stress-mediated activation of JNK and caspase-12 and subsequent activation of mitochondria-dependent caspase cascade regulated by Bcl-xL

Exposure of Jurkat T cells to mollugin (15–30 μM), purified from the roots of Rubia cordifolia L., caused cytotoxicity and apoptotic DNA fragmentation along with mitochondrial membrane potential disruption, mitochondrial cytochrome c release, phosphorylation of c-Jun N-terminal kinase (JNK), activation of caspase-12, -9, -7, -3, and -8, cleavage of FLIP and Bid, and PARP degradation, without accompanying necrosis. While these mollugin-induced cytotoxicity and apoptotic events including activation of caspase-8 and mitochondria-dependent activation of caspase cascade were completely prevented by overexpression of Bcl-xL, the activation of JNK and caspase-12 was prevented to much lesser extent. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk), the caspase-3 inhibitor (z-DEVD-fmk) or the caspase-12 inhibitor (z-ATAD-fmk) at the minimal concentration to prevent mollugin-induced apoptosis appeared to completely block the activation of caspase-7 and -8, and PARP degradation, but failed to block the activation of caspase-9 and -3 with allowing a slight enhancement in the level of JNK phosphorylation. Both FADD-positive wild-type Jurkat clone A3 and FADD-deficient Jurkat clone I2.1 exhibited a similar susceptibility to the cytotoxicity of mollugin, excluding involvement of Fas/FasL system in triggering mollugin-induced apoptosis. Normal peripheral T cells were more refractory to the cytotoxicity of mollugin than were Jurkat T cells. These results demonstrated that mollugin-induced cytotoxicity in Jurkat T cells was mainly attributable to apoptosis provoked via endoplasmic reticulum (ER) stress-mediated activation of JNK and caspase-12, and subsequent mitochondria-dependent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-xL.     

牛乳铁蛋白素对Jurkat细胞和HFL-I细胞生物学特性的影响

目的形态学观察牛乳铁蛋白素(LfcinB)对Jurkat细胞与人胚肺成纤维(HFL-I)细胞作用的程度,验证LfcinB诱导Jurkat细胞凋亡信号转导通路。方法用荧光显微镜观察Hoechst33258染色LfcinB处理过的Jurkat细胞和正常成纤维细胞,与阴、阳性对照组比较;用DNA断裂琼脂糖凝胶电泳分析LfcinB对Jurkat和HFL-I细胞作用差异;用流式细胞术分析Jurkat细胞早期凋亡和线粒体电势电位状况;Westernblotting研究Jurkat细胞接触LfcinB4、6和8h细胞内caspase-3、caspase-8、caspase-9和胞浆中细胞色素C含量的变化。结果在LfcinB作用Jurkat细胞DNA断裂琼脂糖凝胶电泳图中显示为阶梯状凋亡DNALadder,而LfcinB作用的HFL-I细胞无此现象;在荧光显微镜可以看到LfcinB作用过的Jurkat细胞经Hoechst33258染色细胞核呈致密浓染和HFL-I细胞的细胞核呈均匀染色;流式术分析结果为LfcinB作用Jurkat细胞2、4h的凋亡率分别为11.5%和17.8%,线粒体膜电位下降;caspase-3、caspase-9和细胞色素C的免疫印记分析显示,LfcinB能使Jurkat细胞胞浆中的细胞色素C含量增加,被激活caspase-3、caspase-9在胞浆中逐渐增多,对caspase-8没有影响。结论LfcinB诱导Jurkat细胞凋亡,对正常成纤维细胞没有作用;Jurkat细胞接触LfcinB2h以后细胞内caspase-3、caspase-9和胞浆中细胞色素C的含量累积增加,验证了LfcinB通过依赖caspase家族的细胞内信号通路诱导Jurkat细胞凋亡。     

衰变加速因子及其所在膜微区在T淋巴细胞信号传递中的作用

低温抽提Jurkat细胞膜去污剂抗性的膜成分(DIG),检测Src家族酪氨酸激酶(PTK)及衰变加速因子(DAF)的分布情况。共聚焦扫描显微技术检测Jurkat细胞静息与交联状态下DAF分子对去污剂Triton-X100的抗溶性,以探讨其在Jur-kat细胞免疫识别中的作用,结果显示静息状态CD3对去污剂无抗溶性,而DAF与Lck有抗溶性。单抗交联后CD3对去污剂抗性有所增加。静息状态DAF与Src家族PTK特异分布于膜微区中,交联CD3可诱导CD3与膜微区特异性聚集,DAF所在的膜微区可能促进T细胞的免疫识别和信号传递作用。     

在不同应激状态下MDM2基因对Jurkat细胞的作用

[目的]探讨MDM2基因在不同应激状态下对T细胞性白血病细胞株Jurkat增殖、凋亡和基因表达的影响.[方法]采用RNA干扰下调白血病细胞株Jurkat MDM2基因表达后,用Western blot、流式细胞术等方法检测Jurkat细胞在不同应激状态下的变化.[结果]无应激状态下,MDM2基因表达下降后,Jurkat细胞增殖减慢,细胞周期表现一定程度的G1-S阻滞;E2F1表达明显降低超过78%,p65表达下调超过58%;细胞损伤后,MDM2表达下调可导致细胞损伤修复减慢,增强紫外线和甲氨喋呤诱导凋亡作用,导致甲氨喋呤作用下Rb和Bax基因的表达相对增加.[结论]在不同应激状态下,MDM2基因表达下降广泛影响Jurkat细胞的增殖、凋亡和基因表达等生物学特性,提示MDM2基因有可能作为T细胞性白血病治疗的潜在新靶点.     

转录因子Sp1和Sp3对Jurkat T细胞端粒酶活性的调节作用

目的探讨转录因子Sp1、Sp3对JurkatT细胞端粒酶活性和端粒酶逆转录酶(hTERT)的调节作用.方法用脂质体将Sp1、Sp3表达载体转入Jurkat T细胞,用Westem blotting方法检测蛋白表达水平;用端粒酶PCR-ELSA法检测细胞端粒酶活性并用银染显示端粒酶活性梯度;用RT-PCR方法检测hTERTmRNA水平.结果Sp1、Sp3载体转化36 h的细胞Sp1、Sp3蛋白水平分别升高59.6%(P〈0.01)和36.8%(P〈0.05);随着Sp1表达水平的增加,端粒酶活性和hTERT mRNA水平明显增加,增加率分别为38.5%(P〈0.05)和25.4%(P〈0.05).而Sp3表达载体对端粒酶活性和hTERT mRNA水平无明显影响.结论转录因子Sp1通过调节hTERT mRNA转录水平而调节Jurkat T细胞端粒酶活性,Sp3无此作用.     

Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis

Wild-type (wt) p53-induced phosphatase 1 (Wip1), encoded by the protein phosphatase, Mg²⁺/Mn²⁺ dependent 1D (PPM1D) gene, is a serine/threonine phosphatase induced upon genotoxic stress in a p53-dependent manner. Wip1/PPM1D is frequently overexpressed, amplified and mutated in human solid tumors harboring wt p53 and is thus currently recognized as an oncogene. Oncogenic Wip1 dampens cellular stress responses, such as cell cycle checkpoints, apoptosis and senescence, and consequently increases resistance to anticancer therapeutics. Targeting Wip1 has emerged as a therapeutic strategy for tumors harboring wt p53. However, little is known about the efficacy of Wip1-targeted therapies in tumors lacking p53. The present study aimed to investigate the potential role of oncogenic Wip1 in p53 mutant (mt) Jurkat cells. In the present study, it was demonstrated that p53 mt Jurkat cells exhibited PPM1D/Wip1 gene amplification and expressed relatively high levels of Wip1, as confirmed by gene copy number and RNA expression analysis. In addition, Jurkat cells underwent G2 cell cycle arrest, apoptotic cell death and senescence in response to etoposide and doxorubicin, although the phosphorylation levels of DNA damage response (DDR) elements, including ataxia-telangiectasia mutated, ataxia-telangiestasia and Rad3-related, checkpoint kinase (Chk)1 and Chk2 were significantly low. Accordingly, the targeting of Wip1 phosphatase by RNA interference increased the phosphorylation of DDR elements, but decreased the rate of apoptosis in response to etoposide or doxorubicin in Jurkat cells. The induction of senescence or cell cycle arrest was not affected by the knockdown of Wip1. The results suggest that increased Wip1 expression enhances the apoptotic sensitivity of Jurkat cells in response to chemotherapeutic agents by attenuating DDR signaling. The present study highlights the possible pro-apoptotic role of Wip1 in a p53 mt T-cell acute lymphoblastic leukemia cell line. The data suggest the careful consideration of future treatment strategies aiming to manipulate or target Wip1 in human cancers lacking p53.     

Concomitant overexpression of mir‐182‐5p and mir‐182‐3p raises the possibility of IL‐17–producing Treg formation in breast cancer by targeting CD3d,ITK, FOXO1, and NFATs: A meta‐analysis and experimental study

T cells are polarized toward regulatory T cells (Tregs) in tumor microenvironment by the shuttling of microRNAs that target T cell–activating signaling pathways. We evaluated the expression of the miR‐182 cluster (miR‐96, 182, and 183) in peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC), and T cell polarization by the expression of FOXO1, NFATs, ITK, TCR/CD3 complex, and IL‐2/IL‐2RA. Twenty‐six microRNAs overexpressed in tumor tissues and sera of these patients were extracted by a meta‐analysis. Then, the expression of the miR‐182 cluster was investigated in PBMCs and sera of these patients and correlated with their targets in PBMCs. Finally, miR‐182 was cloned into Jurkat cells to evaluate its effects on T cell polarization. FOXO1, CD3d, ITK, NFATc3, NFATc4, and IL‐2RA were targeted by miR‐182, due to which their expression decreased in PBMCs of patients. Although IL‐6, IL‐17, and TGF‐β increased after miR‐182 transduction, IL‐2 dramatically decreased. We revealed CD4⁺FOXP3⁺ T cell differentiation in the miR‐182–transduced group. Although miR‐182 has inhibitory effects on T cells by the inhibition of FOXO1, TCR/CD3 complex, NFATs, and IL‐2/IL‐2RA signaling pathways, it increases FOXP3, TGF‐β, and IL‐17 expression to possibly drive T cell deviation toward the transitional state of IL‐17–producing Tregs and Treg formation in the end.     

CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells

Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4⁺ T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data indicate that CD147 and CD98 might play important roles in cyclophilin-induced cell migration.     

Molecular effects of the phosphatidylinositol-3-kinase inhibitor NVP-BKM120 on T and B-cell acute lymphoblastic leukaemia

BackgroundConstitutive activation of the PI3K pathway in T cell acute lymphoblastic leukaemia (T-ALL) has been reported and in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. We investigated the effects of NVP-BKM120, a potent pan-class I PI3K inhibitor, in lymphoblastic leukaemia cell lines.MethodsEffects of NVP-BKM120 on cell viability, clonogenicity, apoptosis, cell cycle, cell signalling and autophagy were assessed in vitro on T-ALL (Jurkat and MOLT-4) and BL (Daudi and NAMALWA) cell lines.ResultsNVP-BKM120 treatment decreased cell viability and clonogenic growth in all tested cells. Moreover, the drug arrested cell cycling in association with a decrease in Cyclin B1 protein levels, and increased apoptosis. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, mTOR, P70S6K and 4EBP1, with stable total protein levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, in association with augmented BAX:BCL2 ratio. Quantification of autophagy showed a dose-dependent increase in acidic vesicular organelles in all cells tested.ConclusionIn summary, our present study establishes that NVP-BKM120 presents an effective antitumour activity against T-ALL and BL cell lines.