Increased Antigen Presentation but Impaired T Cells Priming after Upregulation of Interferon-Beta Induced by Lipopolysaccharides Is Mediated by Upregulation of B7H1 and GITRL

Dendritic cells are able to present Ag-derived peptides on MHC class I and II molecules and induce T cells priming. Lipopolysaccharides (LPS), an activator of Toll-like 4 receptor (TLR4) signaling, has been demonstrated to facilitate Ag-presentation, up-regulate surface molecules expression but impair T cells priming. In this study, we investigated the effect of LPS on nicotine-enhanced DCs-dependent T cells priming and the mechanisms of LPS orchestrating the immunosuppressive program. We could demonstrate that the treatment with LPS resulted in increased surface molecules expression, enhanced Ag-presentation, up-regulated release of TGF-beta, TNF-alpha, IL-6, and IFN-beta. Concomititantly, the upregulation of IFN-beta in DCs induces the up-regulation of coinhibitory molecules B7H1 and GITRL, which cause an impaired activation of naïve Ag-specific T cells and the induction of T cell tolerance by enhancing B7H1-PD-1 interactions and promoting GITRL-GITL facilitated Treg generation, respectively. These data provide a mechanistic basis for the immunomodulatory action of IFN-beta which might open new possibilities in the development of therapeutic approaches aimed at the control of excessive immune response and persistent infection.     

Upregulation of Daxx mediates apoptosis in response to oxidative stress

Oxidative stress induces apoptosis in a variety of cell types by as yet unclear signaling mechanisms. The Daxx protein is reportedly involved in apoptosis through its interactions with Fas, transforming growth factor-beta receptor, and promyelocytic leukemia protein (PML). Here, we explored the possible roles of Daxx in oxidative stress-induced apoptosis. We found that both the mRNA and protein levels of Daxx markedly increased when cells underwent apoptosis after H2O2 treatment. Pretreatment with the cell-permeable antioxidant, N-acetyl cysteine, prevented cells from H2O2-induced Daxx upregulation and subsequent apoptosis, indicating that the endogenous oxidant regulated Daxx expression. Furthermore, suppression of endogenous Daxx expression by antisense oligonucleotide technology inhibited oxidative stress-induced apoptosis in HeLa cells. Taken together, these results suggest that Daxx acts as an intermediary messenger of pro-apoptotic signals triggered by oxidative stress.     

Application of Freeze-Etching Method to the Study of Reovirus-Infected LLC-MK2 Cells

A continuous cell line of rhesus monkey kidney cells, LLC-MK(2), was infected with reovirus type 1 (Lang). The cells were freeze-etched as a method for observing the structural details of the reovirus-induced cytoplasmic inclusion. Information on maturation may be obtained by preparing infected cells for electron microscopy by freeze-etching.     

Daxx过表达对氧化应激诱导的肝肿瘤细胞凋亡的影响

死亡结构域相关蛋白Daxx可以敏化多种肿瘤细胞的凋亡过程,但对于肝肿瘤细胞株HepG2的影响未见报道．为了研究Daxx增加肝HepG2细胞对药物敏感性的影响及机制,为开发药物新的药理作用提供理论依据,分别转染pEGFP-C1和pEGFP-C1-Daxx这两个载体到HepG2细胞．实验分组如下：（1）正常对照组（未转染细胞组）;（2）pEGFP-C1空载体转染组（HepG2/GFP细胞）;（3）pEGFP-C1-Daxx表达载体转染组（nepG2/GFP-Daxx细胞）．筛选稳定细胞株,用逆转录聚合酶链反应检测mRNA的表达;用过氧化氢孵育24h诱导细胞凋亡,采用MTT法和流式细胞术检测细胞凋亡率,Western blot检测蛋白质的表达．经G418筛选稳定的细胞运用RT-PCR技术分析其mRNA,结果显示,转染绿色荧光蛋白Daxx表达载体的细胞Daxx的mRNA明显上调：用荧光显微镜观察到Daxx蛋白主要定位于细胞核．用过氧化氢诱导HepG2细胞凋亡,观察到过氧化氢呈浓度依赖性地抑制HepG2细胞活性．正常对照细胞、HepG2／GFP、HepG2／GFP-Daxx 3组细胞的IC50值分别是0．72、0．76、0．49mmol／L．并且运用流式细胞仪检测到HepG2／GFP-Daxx组细胞凋亡率明显高于转染空载体质粒组与未转染组（（42．9±8．42）vs（27．3±6．38）or（28．5±4．71））．提示HepG2/GFP-Daxx细胞对过氧化氢的反应性较未转染细胞和HepG2/GFP敏感．还运用Western-blot检测到活化的caspase3在Daxx转染组细胞表达最强,达到（204．66±19．68）％,而未转染和HepG2/GFP组细胞分别是（100±3．1）％、（107．39±20．1）％,进一步说明了Daxx可以增加HepG2细胞对于过氧化氢的敏感性．同时,观察到过氧化氢处理24h后,Daxx转染组细胞磷酸化的JNK表达明显高于空载体转染组和未转染细胞组．上述结果表明：a．Daxx可以增加肝HepG2细胞对过氧化氢诱导的细胞凋亡敏感性;b．Daxx蛋白敏化过氧化氢诱导的HepG2细胞凋亡可能与协同增加JNK活性有关．     

The Hydrogel Nature of Mammalian Cytoplasm Contributes to Osmosensing and Extracellular pH Sensing

Cytoplasm is thought to have many hydrogel-like characteristics, including the ability to absorb large amounts of water and change volume in response to alterations in external environment, as well as having limited leakage of ions and proteins. Some gel-like behaviors have not been rigorously confirmed in mammalian cells, and others should be examined under conditions where gel volume can be accurately monitored. Thus, possible contributions of cytoplasm hydrogel properties to cellular processes such as volume sensing and regulation remain unclear. We used three-dimensional imaging to measure volume of single substrate-attached cells after permeabilization of their plasma membrane. Permeabilized cells swelled or shrinked reversibly in response to variations of external osmolality. Volume changes were 3.7-fold greater than observed with intact cells, consistent with cytoplasm's high water-absorbing capacity. Volume was maximal at neutral pH and shrunk at acidic or alkaline pH, consistent with pH-dependent changes of protein charge density and repulsive forces within cellular matrix. Volume shrunk with increased Mg²⁺ concentration, as expected for increased charge screening and ionic crosslinking effects. Findings demonstrate that mammalian cytoplasm resembles hydrogel and functions as a highly sensitive osmosensor and extracellular pH sensor. Its high water-absorbing capacity may allow rapid modulation of local fluidity, macromolecular crowding, and activity of intracellular environment.     

Rab7 Activation by Growth Factor Withdrawal Contributes to the Induction of Apoptosis

The Rab7 GTPase promotes membrane fusion reactions between late endosomes and lysosomes. In previous studies, we demonstrated that Rab7 inactivation blocks growth factor withdrawal-induced cell death. These results led us to hypothesize that growth factor withdrawal activates Rab7. Here, we show that growth factor deprivation increased both the fraction of Rab7 that was associated with cellular membranes and the percentage of Rab7 bound to guanosine triphosphate (GTP). Moreover, expressing a constitutively GTP-bound mutant of Rab7, Rab7-Q67L, was sufficient to trigger cell death even in the presence of growth factors. This activated Rab7 mutant was also able to reverse the growth factor-independent cell survival conferred by protein kinase C (PKC) δ inhibition. PKCδ is one of the most highly induced proteins after growth factor withdrawal and contributes to the induction of apoptosis. To evaluate whether PKCδ regulates Rab7, we first examined lysosomal morphology in cells with reduced PKCδ activity. Consistent with a potential role as a Rab7 activator, blocking PKCδ function caused profound lysosomal fragmentation comparable to that observed when Rab7 was directly inhibited. Interestingly, PKCδ inhibition fragmented the lysosome without decreasing Rab7-GTP levels. Taken together, these results suggest that Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis and that Rab7-dependent fusion reactions may be targeted by signaling pathways that limit growth factor-independent cell survival.     

铁蛋白介导细胞凋亡的作用机制

铁蛋白是生物体广泛存在且高度保守的可溶性蛋白质,在铁离子稳态维持、胚胎发育调控、细胞增殖以及细胞凋亡等过程中具有重要作用。过量的铁离子能通过芬顿反应产生活性氧,过量的活性氧会造成氧化应激并直接损害DNA、脂质和蛋白质,最终导致细胞凋亡。铁蛋白能够螯合铁离子,进而保护细胞免受氧化应激诱导的细胞凋亡。铁蛋白表达受阻时,细胞内不稳定铁水平升高并诱导氧化应激,最终造成细胞凋亡。同时,氧化应激可在转录和翻译水平调节铁蛋白表达,升高的铁蛋白则参与维持机体氧化还原水平的稳定。本文主要从线粒体途径和死亡受体途径阐明铁蛋白介导细胞凋亡的分子机制,为深入研究铁蛋白功能以及相关疾病治疗提供理论支持。     

高糖通过线粒体途径诱导成骨细胞凋亡

线粒体途径是细胞凋亡的重要途径之一. 在特定的刺激下,例如高糖条件,可以通过caspase依赖性和非依赖性两种途径诱导多种细胞凋亡.但线粒体凋亡途径在高糖引起成骨细胞凋亡中所起的作用,目前尚不清楚.本研究证明,高糖可以通过线粒体凋亡途径诱导成骨细胞凋亡.Annexin V-FITC/PI流式细胞学检测显示,高糖可诱导MC3T3-E1细胞凋亡.Western印迹检测发现,不同浓度D-葡萄糖（11,22,33 mmol/L）可以引起线粒体中Bax蛋白表达的增加,使Bax蛋白由细胞质中易位到线粒体,激活了线粒体凋亡途径.JC-1荧光探针检测证实,高糖处理成骨细胞后,线粒体膜电位明显降低,表明线粒体途径被激活.而细胞质中的细胞色素c、凋亡诱导因子（AIF）表达增加,细胞色素c和AIF从线粒体中释放到细胞质中,释放到细胞质中的细胞色素c使caspase-3、caspase-9剪切活化,从而激活了caspase依赖性凋亡途径.因此,线粒体凋亡途径可能是高糖诱导成骨细胞凋亡过程中一个重要的途径.     

腺病毒介导的人ING4基因诱导C6鼠胶质瘤细胞凋亡

目的：研究肿瘤抑制基因人ING4 (inhibitor of growth family, member 4)对C6鼠胶质瘤细胞的促凋亡作用。方法：将携有绿色荧光蛋白（GFP）腺病毒空载体Ad及重组腺病毒Ad-hING4-His（由本科室构建）分别感染C6细胞,RT-PCR法检测hING4的转录,Western-blotting法检测目的蛋白的表达。并观测hING4基因表达对C6胶质瘤细胞的作用,用MTT法绘制生长曲线,计算抑瘤率。再取重组腺病毒Ad-hING4-His及空腺病毒Ad作用后的C6细胞分别行激光共聚焦显微镜观察凋亡小体、透射电镜观察亚细胞结构的变化,抽提基因组DNA行琼脂糖凝胶电泳及流式细胞仪检测。结果： Ad-hING4-His感染C6细胞后,RT-PCR及Western-blotting结果提示有目的基因的转录和表达。hING4基因表达可以显着抑制C6细胞生长。激光共聚焦观察可见明显核断裂、透射电镜可见实验组细胞呈凋亡表现、基因组DNA电泳呈现梯形条带,流式细胞仪检测有明显AP峰,凋亡率达18.1％。结论：hING4可以通过促进细胞凋亡作用而显着抑制C6细胞的增殖和生长。     

增加bax基因表达促进紫杉醇诱导的食管癌细胞凋亡

为促进紫杉醇诱导的食管癌细胞凋亡,探索克服耐药、提高紫杉醇疗效的新途径 ,构建了ｂａｘ基因可诱导的真核表达载体,并用荧光素酶基因系统测定也金属硫蛋白启动子在食管癌细胞中的诱导表达及控制能力。将ｂａｘ基因转入食管癌细胞Ｅｃａ１０９中,用免疫细胞化学方法测定了Ｂａｘ蛋白表达,并以ＴＵＮＥＬ、ＤＮＡ梯带、流式细胞术等方法分析了转染ｂａｘ对紫杉醇的诱导细胞凋亡的促进作用。结果表明,１４０μｍｏｌ／ＬＺｎｓ     

Both STING and MAVS Fish Orthologs Contribute to the Induction of Interferon Mediated by RIG-I

Viral infections are detected in most cases by the host innate immune system through pattern-recognition receptors (PRR), the sensors for pathogen-associated molecular patterns (PAMPs), which induce the production of cytokines, such as type I interferons (IFN). Recent identification in mammalian and teleost fish of cytoplasmic viral RNA sensors, RIG-I-like receptors (RLRs), and their mitochondrial adaptor: the mitochondrial antiviral signaling (MAVS) protein, also called IPS-1, highlight their important role in the induction of IFN at the early stage of a virus infection. More recently, an endoplasmic reticulum (ER) adaptor: the stimulator of interferon genes (STING) protein, also called MITA, ERIS and MPYS, has been shown to play a pivotal role in response to both non-self-cytosolic RNA and dsDNA. In this study, we cloned STING cDNAs from zebrafish and showed that it was an ortholog to mammalian STING. We demonstrated that overexpression of this ER protein in fish cells led to a constitutive induction of IFN and interferon-stimulated genes (ISGs). STING-overexpressing cells were almost fully protected against RNA virus infection with a strong inhibition of both DNA and RNA virus replication. In addition, we found that together with MAVS, STING was an important player in the RIG-I IFN-inducing pathway. This report provides the demonstration that teleost fish possess a functional RLR pathway in which MAVS and STING are downstream signaling molecules of RIG-I. The Sequences presented in this article have been submitted to GenBank under accession numbers: Zebrafish STING ({"type":"entrez-nucleotide","attrs":{"text":"HE856619","term_id":"410810118","term_text":"HE856619"}}HE856619); EPC STING ({"type":"entrez-nucleotide","attrs":{"text":"HE856620","term_id":"410810120","term_text":"HE856620"}}HE856620); EPC IRF3 ({"type":"entrez-nucleotide","attrs":{"text":"HE856621","term_id":"410810122","term_text":"HE856621"}}HE856621); EPC IFN promoter ({"type":"entrez-nucleotide","attrs":{"text":"HE856618","term_id":"410810117","term_text":"HE856618"}}HE856618).     

Sodium Selenite-Induced Apoptosis Mediated by ROS Attack in Human Osteosarcoma U2OS Cells

Sodium selenite (Na₂SeO₃, SSE) is an inorganic Se compound that is widely used in cancer chemoprevention studies. SSE has been shown to have anti-proliferative effects on several types of human cancer cells, but its effect on osteosarcoma cells has thus far not been reported. In this study, the cytotoxic effect of SSE on osteosarcoma cells U2OS was investigated in vitro and found to be higher than on comparable non-cancer cell lines 293 and L6. Treatment with SSE decreased cell growth in a dose- and time-dependent manner and altered cellular morphology. SSE also inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and accumulation of cells during the advanced phase of apoptosis. SSE-induced apoptosis correlated with the activation of CASP 3, downregulation of BCL-2, and upregulation of P53 and PTEN in U2OS cells. These results indicated that SSE induces apoptosis in U2OS cells mainly through an ROS-mediated caspase pathway. This is the first report to show a possible mechanism of the anti-proliferative effect of SSE for the prevention of osteosarcoma in cell culture models.     

VCAM-1 Upregulation Contributes to Insensitivity of Vemurafenib in BRAF-Mutant Thyroid Cancer

Vemurafenib, an inhibitor of mutant BRAF activity, is a promising anticancer agent for patients with BRAF-mutant metastatic melanoma. However, it is less effective in BRAF-mutant thyroid cancer, and the reason for this discrepancy is not yet fully elucidated. By RNA sequencing analysis, we identified vascular cell adhesion molecular-1 (VCAM-1) to be highly upregulated in both time- and dose-dependent manners during BRAF inhibition (BRAFi) in a BRAF-mutant papillary thyroid cancer cell line (BCPAP). Cell cytotoxicity and apoptosis assays showed that knockdown of the induced VCAM-1 in BCPAP cells augmented the antitumor effects of vemurafenib, with decreased IC50 values of 1.4 to 0.8 μM. Meanwhile, overexpression of VCAM-1 in a BRAF-mutant anaplastic thyroid cancer cell line (FRO) reduced the sensitivity to vemurafenib, with increased IC50 values of 1.9 to 5.8 μM. Further investigation showed that PI3K-Akt-mTOR pathway was activated during BRAFi. Co-treatment with Akt signaling inhibitor MK2206 decreased the induced expression of VCAM-1 during BRAFi. This combination further improved the efficacy of vemurafenib. Moreover, VCAM-1 promoted migration and invasion in thyroid cancer cells in vitro, which was also indicated in thyroid cancer patients. The present study is the first to demonstrate that VCAM-1 is upregulated in thyroid cancer cells treated with vemurafenib and contributes to vemurafenib resistance in BRAF-mutant thyroid cancer cells. Targeting the PI3K-Akt-mTOR pathway–mediated VCAM-1 response may be an alternative strategy to sensitize BRAF-mutant thyroid cancers to vemurafenib.