Metabolomic Profiling of Drug Responses in Acute Myeloid Leukaemia Cell Lines

Combined bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) exert unexpected antileukaemic activities against acute myeloid leukaemia (AML) and these activities are associated with the generation of reactive oxygen species (ROS) within the tumor cells. Although the generation of ROS by these drugs is supported by preceding studies including our own, the interrelationship between the cellular effects of the drugs and ROS generation is not well understood. Here we report the use of NMR metabolomic profiling to further study the effect of BEZ and MPA on three AML cell lines and to shed light on the underlying mechanism of action. For this we focused on drug effects induced during the initial 24 hours of treatment prior to the onset of overt cellular responses and examined these in the context of basal differences in metabolic profiles between the cell lines. Despite their ultimately profound cellular effects, the early changes in metabolic profiles engendered by these drugs were less pronounced than the constitutive metabolic differences between cell types. Nonetheless, drug treatments engendered common metabolic changes, most markedly in the response to the combination of BEZ and MPA. These responses included changes to TCA cycle intermediates consistent with recently identified chemical actions of ROS. Notable amongst these was the conversion of α-ketoglutarate to succinate which was recapitulated by the treatment of cell extracts with exogenous hydrogen peroxide. These findings indicate that the actions of combined BEZ and MPA against AML cells are indeed mediated downstream of the generation of ROS rather than some hitherto unsuspected mechanism. Moreover, our findings demonstrate that metabolite profiles represent highly sensitive markers for genomic differences between cells and their responses to external stimuli. This opens new perspectives to use metabolic profiling as a tool to study the rational redeployment of drugs in new disease settings.     

工程细胞单克隆筛选及单克隆源性验证

工程细胞的单克隆源性是保证产品质量的重要因素之一,因此得到了越来越多的重视。当前,药物审评机构要求报批单位采用合适的实验手段证明所使用细胞的单克隆源性。综述通过介绍生产用工程细胞株单克隆的挑选过程及诸如有限稀释法、ClonePix、流式细胞术等常用方法,讨论如何确保工程细胞的单克隆源性,以及在生物药品生产过程中保证工程细胞单克隆源性的意义。     

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

Background

The increased sequencing of pathogen genomes and the subsequent availability of genome-scale functional datasets are expected to guide the experimental work necessary for target-based drug discovery. However, a major bottleneck in this has been the difficulty of capturing and integrating relevant information in an easily accessible format for identifying and prioritizing potential targets. The open-access resource TDRtargets.org facilitates drug target prioritization for major tropical disease pathogens such as the mycobacteria Mycobacterium leprae and Mycobacterium tuberculosis; the kinetoplastid protozoans Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi; the apicomplexan protozoans Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii; and the helminths Brugia malayi and Schistosoma mansoni.

Methodology/Principal Findings

Here we present strategies to prioritize pathogen proteins based on whether their properties meet criteria considered desirable in a drug target. These criteria are based upon both sequence-derived information (e.g., molecular mass) and functional data on expression, essentiality, phenotypes, metabolic pathways, assayability, and druggability. This approach also highlights the fact that data for many relevant criteria are lacking in less-studied pathogens (e.g., helminths), and we demonstrate how this can be partially overcome by mapping data from homologous genes in well-studied organisms. We also show how individual users can easily upload external datasets and integrate them with existing data in TDRtargets.org to generate highly customized ranked lists of potential targets.

Conclusions/Significance

Using the datasets and the tools available in TDRtargets.org, we have generated illustrative lists of potential drug targets in seven tropical disease pathogens. While these lists are broadly consistent with the research community''s current interest in certain specific proteins, and suggest novel target candidates that may merit further study, the lists can easily be modified in a user-specific manner, either by adjusting the weights for chosen criteria or by changing the criteria that are included.     

Clonal Variation In Proliferation Rate of Cultures of Gpk Cells

Pedigrees of twenty-six clones of a line of keratocytes derived from guinea-pig ear epidermis (GPK cells) were analysed from time-lapse film. the mean interdivision time (IDT) for the culture was 1143 φ 215 (SD) min. the mean generation rates (mean reciprocal interdivision times) of clones varied over a range of 3.93–10.2 × 10^-4/min and the standard deviation of the clonal mean generation rates was 16.8% of the average value. Transient intraclonal variations in IDT due to mitoses in a plane perpendicular to the substratum were observed. the data were also analysed on the basis of cell location in sixteen equal zones (quadrats) of the filmed area. the mean generation rate of quadrats was 8.73 x 10^-4/min (SD = 4.9%). the spatial distribution showed some clustering of cells. the mean local density of the clones (2.25 φ 0.62 cells/10^-4 cm²) was significantly higher than the quadrat density (1.76 φ 0.8 cells/10^-4 cm²). There was no significant correlation between clonal density and mean generation rates, whereas for quadrats a significant negative correlation was found (P= 2.7%). the results support the proposition that cell lineage is the major determinant of the proliferation rate of subconfluent cultures.     

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the ‘mast cell knock-in’ approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.     

Addition of Valproic Acid to CHO Cell Fed-Batch Cultures Improves Monoclonal Antibody Titers

Improving the productivity of a biopharmaceutical Chinese hamster ovary (CHO) fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. One method for increasing CHO cell productivity is the addition of histone deacetylase (HDAC) inhibitors to the cell culture process. In this study, we examined the effect of valproic acid (VPA, 2-propylpentanoic acid), a branched-chain carboxylic acid HDAC inhibitor, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. We then performed factorial experiments to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, we performed verification experiments in fed-batch bioreactors for cell lines A and B. These experiments confirmed that a high dose of VPA late in the culture can increase harvest titer >20 % without greatly changing antibody aggregation, charge heterogeneity, and N-linked glycosylation profiles. Our results suggest that VPA is an attractive and viable small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.     

Retention of Differentiated Function in Clonal Animal Cell Lines, Particularly Hormone-Secreting Cultures

Clonal hormone-secreting cell lines have been established fromanimal tumors. These lines include adrenal steroid-secretingcells, growth hormone-secreting cells, steroid-secreting Leydigcells, and ACTH-secreting cells that retain their differentiatedfunction for prolonged periods in continuous culture. In addition,fibroblast cell lines that secrete a material which stimulatessteroidogenesis by adrenal cells and Leydig cells are described.A systematic approach to obtain functional cultures and thegeneral problem of retention of differentiated function in culturedcells are discussed.     

通过共表达转铁蛋白和细胞周期蛋白D1改造CHO细胞株

目的:对现有的CHO-DG44细胞株进行改造,得到在无血清培养基中更具生长优势的CHO宿主细胞株。方法:分别克隆转铁蛋白和细胞周期蛋白D1基因,构建共表达2种基因的pIRES质粒,转染CHO-DG44细胞株,筛选G418抗性克隆。结果与结论:得到3株G418阳性克隆株,其中转铁蛋白和细胞周期蛋白D1表达水平最高的S6与CHO-DG44相比,在无血清培养基中生长更快、密度更高。     

水稻重复序列RRD3在CHO和3T3细胞中的启动子活性

RRD3是通过变性 -复性动力学从水稻基因组中克隆到的一个中度重复序列 .将 RRD3作为启动子亚克隆到氯霉素乙酰基转移酶基因为报告基因的 p CAT载体上 ,转染 5种动物细胞 .结果表明在 CHO、3T3细胞中能够启动 CAT基因的表达 .利用核酸外切酶 和 PCR构建了 RRD3的系列缺失体 ,经转染后的 CAT活性分析表明 ,该序列中存在多个与哺乳动物细胞中启动基因表达有关的调控元件 .结合 RRD3在大肠杆菌和高等植物烟草中启动报告基因表达的活性 ,对重复序列与生物进化的关系进行了讨论 .     

Replication of Nosema Algerae In Three Insect Cell Lines

SYNOPSIS Nosema algerae , a microsporidan parasite of anopheline mosquitoes, was successfully replicated in 3 insect cell culture lines: Trichoplusia ni (TN-368); Heliothis zea (IPLB-1075); and Mamestra brassicae (IZD-Mb-0503). Infectious spores were produced in vitro. Spores were observed at 48 h postinfection, and some cells were filled with sproes by 72 h.
The number of parasites per cell increased with time. At 72 h postinfection, the infection rates for the 3 cell lines ranged from 23 to 32%. Infected cell lines were subcultured, and by the 6th passage spore production had ceased.     

Karyotype Characterization of In Vivo- and In Vitro-Derived Porcine Parthenogenetic Cell Lines

Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.     

用于生产重组蛋白药物的抗凋亡CHO宿主细胞株的建立

哺乳动物工程细胞在大规模培养生产重组蛋白时很容易发生细胞凋亡,从而导致生产过程提前终止,造成生产成本高昂。细胞代谢产物氨已被证明可以促进细胞凋亡,而线粒体膜整合蛋白Bcl-2可以通过促进线粒体膜完整性而抑制细胞凋亡。本实验应用谷氨酰胺合成酶加压系统在CHO工程细胞中高效表达中国仓鼠Bcl-2蛋白,使细胞具有抗凋亡能力的同时,利用谷氨酸和氨合成谷氨酰胺而有效降低培养基中氨的含量,从而达到抑制细胞凋亡的目的。     

Murine Sarcoma and Leukemia Viruses: Assay Using Clonal Lines of Contact-Inhibited Mouse Cells

A continuous cell line of highly contact-inhibited cells (NIH/3T3) has been developed from NIH Swiss mouse embryo cultures. Its growth properties are similar to those of 3T3 and BALB/3T3. Although 3T3 is relatively insensitive to focus formation by murine sarcoma viruses, cloned lines of both NIH/3T3 and BALB/3T3 have been isolated that are highly sensitive to sarcoma virus focus formation and leukemia virus growth. The sensitivity and specificity are comparable to those found with primary embryo cells. MSV-transformed lines of NIH/3T3 have been obtained.     

起源于组织培养的籼稻雄性不育体细胞无性系变异

IR8及IR54的成熟种子与幼穗培养在MS培养基,蔗糖6％附加生长刺激素及其它成分的条件下,在再分化植株第一代(R_1)中各发现了1株雄性不育突变(其中来自IR54的1株为育性嵌合体)。在IR24及IR54体细胞无性系第二代(R_2)各发现1个株系分离出雄性不育与育性正常两类植株。上述雄性不育有花粉败育与无花粉型两类。IR24对无花粉型的雄性不育(来自IR54)为半恢复。F_1及R_2代的育性分离完全符合15/16∶1/16的模式,表明雄性不育为两对独立的核基因所控制。通常雄性不育是通过杂交(包括远缘与品种间)理化诱变或自发突变产生的。最近在烟草上通过原生质体融合也获得了雄性不育。但从体细胞培养的无性系及其后代中获得雄性不育的突变,本研究乃属首次。