Different host-cell shutoff strategies related to the matrix protein lead to persistence of vesicular stomatitis virus mutants on fibroblast cells

Acute infection of fibroblastic cell lines by the Indiana strain of vesicular stomatitis virus (VSV) usually induces dramatic cytopathic effects and shutoff of cellular gene expression. We have compared a series of independent mutants with differences in shutoff induction and found that M was mutated either in the N-terminus (M(51)R) or C-terminus (V(221)F and S(226)R). Furthermore, only double mutants (M mutation and a ts mutation related or not to M) were able to persist on fibroblast cell lines at 39 degrees C. A more detailed investigation of the infection was performed for the mutants T1026, TP3 and G31, differing in their host shutoff effects related to M protein. Viral activity in persistently infected mouse L-929 and monkey Vero cell lines was followed by viral proteins detection, RNA synthesis throughout infection and finally detection of infectious particles. All three mutants cause extensive CPE followed by emergence of persistently infected cells on Vero cells. The same thing is seen on L-929 cells except for T1026 which causes little CPE. Taken together, the results form a basis of further studies to clarify how various viral and cellular factors interact in the establishment of a persistent infection by VSV mutants.     

Rabbit tumor necrosis factor: mechanism of action.

Rabbit tumor necrosis factor (TNF) was examined for effects on normal and transformed cells in culture. Several assays for killing of L-929 cell targets were developed, and their sensitivities were compared. Normal cells were not killed by TNF, and the discrimination between normal and transformed cells was shown not to be due to a cell cycle-dependent mechanism. TNF killing of L-929 cells was delayed for 10 to 12 h and thereafter showed concentration and time-dependent increases in cytolysis. Actinomycin D or cycloheximide treatment of L-929 cells resulted in an enhancement of the rate of cell killing as well as a shortening of the preceding lag period. TNF killing of L-929 cells was temperature dependent; cells were considerably more resistant to lysis at 25 degrees C and showed enhanced killing at 39 degrees C as compared to 37 degrees C controls. The slope of the dose curve showed less than single-hit kinetics. A model for cell killing whose general features incorporate both the specificity and catalytic properties of an enzymatic reaction is proposed for TNF action.     

Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.     

Changes in gene expression profiles in developing B cells of murine bone marrow.

Gene expression profiles of five consecutive stages of mouse B cell development were generated with high-density oligonucleotide arrays from as few as 2 x 10(4) ex vivo isolated and flow-cytometrically purified cells. Between 2.8% and 6.8% of all genes change on differentiation from one cellular stage to the next by at least twofold. The entire pathway involves differential expression of 10.7% of all genes. Previously known expression patterns of 15 genes (like surrogate light chain, RAG-1/2, MHC class II, mel-14 antigen) are confirmed. The gene expression patterns of the proliferating pre-BI and large pre-BII cells on the one hand, and the resting immature and mature B cells on the other hand, are most similar to each other. Small pre-BII cells display a pattern that is transitional between these two groups. Most of the genes expressed in early precursors are involved in general processes, like protein folding or cell cycle regulation, whereas more mature precursors express genes involved in more specific molecular programs (cell surface receptors, secreted factors, and adhesion molecules, among others). Between 19 and 139 genes share a given expression pattern. Combining knowledge about gene function and expression pattern allows identification of novel candidate genes potentially involved in self-maintenance of pre-BI cells, allelic exclusion and pre-B cell receptor signaling in large pre BII cells, cell-cycle arrest of small pre-BII cells, propensity toward apoptosis or anergization in immature B cells, propensity toward cell division and activation in mature B cells, and stage-specific interactions with stromal cells in the bone marrow.     

Measles virus induces cell-type specific changes in gene expression

Measles virus (MV) causes various responses including the induction of immune responses, transient immunosuppression and establishment of long-lasting immunity. To obtain a comprehensive view of the effects of MV infection on target cells, DNA microarray analyses of two different cell-types were performed. An epithelial (293SLAM; a 293 cell line stably expressing SLAM) and lymphoid (COBL-a) cell line were inoculated with purified wild-type MV. Microarray analyses revealed significant differences in the regulation of cellular gene expression between these two different cells. In 293SLAM cells, upregulation of genes involved in the antiviral response was rapidly induced; in the later stages of infection, this was followed by regulation of many genes across a broad range of functional categories. On the other hand, in COBL-a cells, only a limited set of gene expression profiles was modulated after MV infection. Since it was reported that V protein of MV inhibited the IFN signaling pathway, we performed a microarray analysis using V knockout MV to evaluate V protein's effect on cellular gene expression. The V knockout MV displayed a similar profile to that of parental MV. In particular, in COBL-a cells infected with the virus, no alteration of cellular gene expression, including IFN signaling, was observed. Furthermore, IFN signaling analyzed in vitro was completely suppressed by MV infection in the COBL-a cells. These results reveal that MV induces different cellular responses in a cell-type specific manner. Microarray analyses will provide us useful information about potential mechanisms of MV pathogenesis.     

How adenovirus strives to control cellular gene expression

Host cell gene expression during the course of a human adenovirus infection in synchronized primary human lung fibroblasts was analyzed using cDNA microarrays. The slow progression of the infectious cycle in these cells allowed a detailed examination of cellular gene regulation. In total, 988 unique genes were identified as differentially expressed more than 2-fold. The cellular gene expression profiles closely correlated to the progression of the infection. Based on the observed expression patterns, the deregulation of cellular genes' expression could be separated into four periods: (i) the immediate response of the host to incoming virus; (ii) deregulation of cellular genes involved in cell cycle, growth control, and antiviral response; (iii) steady-state regulation of cellular gene expression during viral DNA replication; (iv) targeting of cellular genes involved in intra- and extra-cellular structure at the late phase of infection. The struggle of the virus to gain control of TGF-beta and Wnt signaling, as well as the apoptotic pathways, was conspicuous.     

K562细胞VEGF基因表达下调后的基因表达谱改变

目的探讨血管内皮细胞生长因子（vascular endothelial growth factor，VEGF）基因VEGF表达下调对白血病K562细胞株基因表达谱的影响。方法采用脂质体介导的方法将抗VEGF发夹状核酶基因真核表达载体pcDNA-RZ转染白血病细胞株K562、G418抗性筛选获得阳性克隆；抽提基因组DNA，用PCR方法验证核酶基因已转入K562细胞；荧光定量PCR和免疫印迹反应检测白血病细胞中VEGF mRNA和蛋白表达量的改变；应用cDNA微阵列技术检测VEGF基因表达下调对白血病K562细胞株基因表达谱的影响。并用逆转录PCR验证PCNA、GSN基因的表达改变。结果抗VEGF发夹状核酶基因真核表达载体pcDNARZ转入白血病细胞株K562、G418筛选两周获得阳性克隆，PCR检测证实核酶基因整合入白血病细胞基因组DNA；与K562及K562／PC细胞（转染空质粒的K562细胞）相比，转染VEGF核酶基因的K562／RZ细胞VEGF mRNA和蛋白的表达量明显降低，芯片中共有表达差异的基因191条，包括周期相关基因、细胞凋亡相关基因、癌基因以及细胞信号和传递蛋白等基因，其中104条表达下调，87条表达上调。逆转录PCR证实GSN基因表达上调，PCNA基因表达下调。结论VEGF基因表达下调能引起白血病K562细胞株基因表达谱的改变，这些基因的改变可能对白血病细胞增殖、分化和凋亡等生物学行为产生了一定的影响。     

In vitro activation of murine bone marrow-derived macrophages with cisplatin and mitomycin-C

Peritoneal macrophages from BALB/c mice after treatment for 24 h in vitro with cisplatin, lipopolysaccharide (LPS) or mitomycin-C were rendered significantly cytotoxic against L-929 tumor target cells. In a similar experiment none of these agents could induce tumoricidal activity of fresh non-adherent bone marrow cells (NABMC). NABMC when incubated in medium alone or in medium containing L-929 culture medium (L-929 CM), a form of macrophage colony stimulating factor (M-CSF), for three days matured to macrophages which were positive for non-specific esterase staining. These bone marrow-derived macrophages cultured with medium alone did not respond to cisplatin. LPS or mitomycin-C for induction of tumoricidal activity whereas bone marrow derived macrophages with that were incubated with L-929 CM showed also significantly enhanced cytotoxicity after treatment with cisplatin, LPS and mitomycin-C. Culturing of NABMC with L-929 CM significantly enhanced cell survival as compared to the cells incubated in medium alone. These results suggest that bone marrow cells not only mature in L-929 CM but also are primed by L-929 CM for induction of tumoricidal activity.     

The immediate early genes of human cytomegalovirus upregulate expression of the cellular genes myc and fos.

Human cytomegalovirus (HCMV) is an important pathogen of the lung. We determined whether the HCMV immediate early genes (IE1 and IE2) can alter the regulation of the cellular immediate early genes (c-fos and c-myc). Plasmid constructs containing the promoter-regulatory regions c-myc or c-fos upstream of the reporter gene, chloramphemicol acetyl transferase, were co-transfected into T cells (Jurkat cells), monocytes/macrophages (THP-1 cells), or human fibroblast cells with plasmid constructs containing the promoter-regulatory region of the HCMV IE genes upstream of the bona fide IE1, IE2 or IE+2 genes; a plasmid that contained no IE coding region was used as a control. These studies show that both products of the HCMV IE genes markedly upregulated expression of the cellular c-fos and c-myc genes. The viral effects of individual proteins (IE1 or IE2) were dependent both on the promoter-regulatory region of the cellular gene and the cell type. In all cells, the combination of IE1 and IE2 further upregulated both cellular genes, suggesting a synergistic effect of IE1 with IE2. Both of the c-myc promoters (P1 and P2) were up-regulated by the HCMV IE gene products. IE1 and IE2 also upregulated the cells' endogenous c-myc and c-fos genes, as determined by amounts of the respective mRNAs. These studies show that HCMV can markedly alter cellular IE gene expression and that the effects of HCMV IE1 and IE2 proteins are dependent both on the promoter-regulatory region of the cellular gene and the type of cell in which the interaction occurs.     

三种贵金属烤瓷合金与高糖对小鼠成纤维细胞L-929增殖的影响

背景：糖尿病患者这一特殊群体口腔环境较为特殊,关于口腔修复材料在这类患者应用的安全性研究较少见报道。 目的：检测3种贵金属烤瓷合金与高糖对体外培养的小鼠成纤维细胞L-929增殖的影响。 方法：将89%金合金、74%金合金、银钯合金3种烤瓷材料浸泡在不同糖浓度(11.1,22.2,33.3 mmol/L)的培养液中浸提,将所得的浸提液与小鼠成纤维细胞L-929共培养。同期设立空白对照。 结果与结论：L-929在不同贵金属烤瓷合金和不同糖浓度的细胞培养液中的增殖活性不同,24,72 h两因素交互作用的P值均小于0.05。与阴性对照组相比,22.2 mmol/L糖浓度下89%金合金促进细胞的增殖(P=0.004),银钯合金则抑制细胞的增殖(P < 0.001),74%金合金在不同糖浓度的培养液中与阴性对照组相比其细胞的增殖活性差异无显著性意义。结果表明,上述3种材料中74%金合金在不同糖浓度下对细胞增殖的影响最小。 关键词：贵金属烤瓷合金;高糖;金合金;银钯合金;小鼠成纤维细胞;增殖 doi:10.3969/j.issn.1673-8225.2012.12.019     

Replication of sialodacryoadenitis virus in mouse L-2 cells

Summary Sialodacryoadenitis (SDA) is a naturally-occurring infection of the laboratory rat raused by the coronavirus, sialodacryoadenitis virus (SDAV). The study of SDAV has been limited because there is no widely available continuous cell line for the propagation of high titers of the virus. The purpose of this study, therefore, was to compare the ability of SDAV to replicate in the permanent cell lines, LBC, of rat origin, and the mouse cell lines. L-929 and L-2. Following 2 to 6 repeated passages of SDAV in LBC cells, the virus could be readily propagated in LBC and L-2 cells, but not in L-929 cells. Similarly, SDAV adapted to replicate directly in L-2 cells could be readily propagated in LBC, but not L-929 cells. In LBC and L-2 cells, cytopathic effect (CPE), viral antigen, viral particles, and virus infectivity could be demonstrated. Titers of up to 10^8.0 infectious viral particles/0.25 ml of culture fluid were obtained at 48 hours in L-2 cells. Titers in LBC cells were one to two logs lower. When susceptible rats were inoculated with eighth passage L-2 cell-adapted virus, they developed typical lesions of SDA. Virus could be recovered from infected tissues and propagated in L-2 cells on first passage. The ability to propagate SDAV to high titers in the widely available L-2 cell line should promote the study of this virus and facilitate its comparison with other murine coronaviruses.     

Bioactive human recombinant tumor necrosis factor-α: an unstable dimer?

As determined by native polyacrylamide gel electrophoresis (PAGE) and gel chromatography the molecular mass of native tumor necrosis factor (TNF)-alpha was approximately 35 kDa. When incubated at low concentrations (less than 1 nM) 125I-labeled TNF-alpha and unlabeled TNF-alpha rapidly multimerized or dissociated into monomers and bioactivity decreased. Sodium dodecyl sulfate (SDS)-PAGE analysis of cross-linked 125I-labeled TNF-alpha demonstrated bands of multi- and trimeric TNF-alpha in addition to dominating bands of dimers and monomers. Tri-, di- and monomeric TNF-alpha were recovered from SDS-PAGE gels and allowed to renature. Of the original receptor-binding activity, 10%-15% was obtained with cross-linked TNF-alpha dimers, whereas none was recovered from preparations of trimeric TNF-alpha. Multimeric and monomeric TNF-alpha exhibited little or no binding activity, and cell-bound, cross-linked TNF-alpha which was dissociated from cellular binding sites was mainly dimeric. 125I-labeled TNF-alpha bound to lymphokine-activated killer (LAK) cells and binding kinetics were much similar (Kd approximately 100 pM) to those reported in other normal cell types. The number of receptors per LAK cell was approximately 4 x 10(3). Cross-linking of TNF-alpha to binding sites in U-937 and LAK cells yielded a receptor-ligand complex of about 80/90 kDa. At 37 degrees C, 125I-labeled TNF-alpha was rapidly internalized and degraded in L-929, U-937 and LAK cells. Degradation of ligand and recycling of receptors were blocked in the presence of methylamine. Methylamine significantly inhibited TNF-alpha-mediated cytolysis of L-929 cells and caused a quantitatively corresponding reduction in cellular TNF-alpha uptake, indicating that L-929 lysis was mediated by receptors.     

通心络对同型半胱氨酸损伤的内皮细胞的基因表达谱的影响

目的: 探讨通心络对同型半胱氨酸(Hcy)损伤的人脐静脉内皮细胞(HUVECs)的基因表达谱的影响。方法: 原代培养的HUVECs,随机分为对照组、Hcy组和通心络组,提取总RNA并纯化,反转录合成荧光分子(Cy3/Cy5)标记的cDNA探针,采用高通量Affymetric人类全基因组寡核苷酸微阵列芯片(含47 000个基因或基因片段)杂交,杂交信号经扫描和数字化处理,通过生物信息学数据分析推测通心络对同型半胱氨酸损伤的HUVECs细胞功能的影响以及可能的信号通路。结果: 与对照组相比,Hcy组有4 343个基因表达上调,316个基因表达下调;与Hcy组相比,通心络组有3 409个基因表达上调,121个基因表达下调。这些共同的差异基因生物功能涉及转录因子调控、激酶、细胞骨架与蛋白合成、转运功能、细胞因子、细胞-细胞受体相互作用和细胞黏附因子等。参与的信号通路主要是与血管新生、凋亡、氧化应激、凝血纤溶和炎症等相关的信号通路,如mTOR信号通路、MAPK信号通路和Toll样受体信号通路。结论: 通心络能导致同型半胱氨酸损伤内皮细胞基因表达谱的改变,这些基因改变可能参与了细胞凋亡、氧化应激和凝血纤溶等过程。     

Author index for volume 132

It has previously been shown that a strain of thymidine kinase (tk)-deficient mouse L-929 cells was unable to respond to murine β-interferon by induction of an anti-viral state and synthesis of double-stranded, RNA-dependent enzymes. Sensitivity to interferon can be restored by introducing into the cells a segment of Herpes simplex virus DNA containing the viral tk gene. It is shown here that not all Ltk(?) cell strains are resistant to interferon, suggesting that expression of a tk gene is not a prerequisite for response to interferon. Introduction of various genes into the resistant Ltk(?) strain, either alone or together with DNA containing the Herpes virus tk gene, leads to restoration of interferon sensitivity only when tk-containing DNA is inserted, showing that the activation of interferon responsiveness is not an artifact of the gene transfer, selection, and cloning procedures. The results imply that a component of the Herpes virus DNA introduced into the cells is able to activate interferon sensitivity.     

Elucidating tumor necrosis factor signaling pathway using a functional gene ddentification approach

Functional identification of genes is an efficient way to study many biological processes in lower eukaryotes. However, an effective approach in mammalian cells is still under development. We designed a functional gene identification procedure and applied it in a study of tumor necrosis factor (TNF)-induced cell killing. This procedure employed a specially designed retroviral vector that allows random truncation of genes, efficiently selecting clones in which a gene was disrupted and quickly identifying disrupted genes. We have identified several novel genes by a preliminary test of this approach and confirmed by reconttitution that the genes we identified are required for TNF cytotoxicity in L929 cells. Because of the efficient identification of these components in TNF-induced cell killing, we have already been able to outline the killing pathway of TNF in L929 cells. Application of this method could be widespread because it can be used in studying any cellular responses if a specific selection assay can be set up.