Queuine hypomodification of tRNA induced by 7-methylguanine

Transfer RNA isolated from Chinese hamster cells transformed by 7-methylguanine is hypomodified for queuine. 7-Methylguanine rapidly induces queuine hypomodification of tRNA in normal Chinese hamster embryo cells under conditions leading to transformation, and the enzyme catalyzing the queuine modification reaction, tRNA: guanine transglycosylase, is inhibited by 7-methylguanine $\text{in}\text{vitro}$.     

Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity

To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL‐sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate (μ), but increased culture longevity and specific mAb productivity (q_mAb) in a dose‐dependent manner. The beneficial effect of valeric acid on culture longevity and q_mAb outweighed its detrimental effect on μ, resulting in 2.9‐fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galactosylation. Unexpectedly, galactosylation of the mAb increased by the 1.5 mM valeric acid addition. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells.     

Filamin A regulates cell spreading and survival via beta1 integrins

Cell spreading and exploration of topographically complex substrates require tightly-regulated interactions between extracellular matrix receptors and the cytoskeleton, but the molecular determinants of these interactions are not defined. We examined whether the actin-binding proteins cortactin, vinculin and filamin A are involved in the formation of the earliest extensions of cells spreading over collagen or poly-L-lysine-coated smooth and beaded substrates. Spreading of human gingival fibroblasts was substantially reduced on beaded or poly-L-lysine-coated substrates. Filamin A, vinculin and cortactin were found in cell extensions on smooth collagen. HEK-293 cells also spread rapidly on smooth collagen and formed numerous cell extensions enriched with filamin A. Knockdown of filamin A in HEK-293 cells by short hairpin RNA reduced spreading and the number of cell extensions. Blocking beta1 integrin function significantly reduced cell spreading and localization of filamin A to cell extensions. Conversely, filamin A-knockdown reduced beta1 integrin-collagen binding as measured by 12G10 antibody, suggesting co-dependence between filamin A and beta1 integrin functions. TUNEL staining showed higher percentages of apoptosis after filamin A-knockdown in spreading cells. Chelation of [Ca2+]i with BAPTA/AM reduced spreading of wild-type and filamin A-knockdown cells, however wild-type cells showed recruitment of filamin A to the subcortex, indicating independent roles of filamin A and [Ca2+]i in cell spreading. We conclude that filamin A integrates with beta1 integrins to mediate cell spreading and prevent apoptosis.     

Combining various strategies to increase the coverage of the plant cell wall glycoproteome

Glycoproteomics recently became a very active field, mostly in mammals. The first part of this paper consists of a mini-review on the strategies used in glycoproteomics, namely methods for enrichment in glycoproteins and mass spectrometry (MS) techniques currently used. In a second part, these strategies are applied to the cell wall glycoproteome of etiolated hypocotyls of Arabidopsis thaliana, showing their complementarity. Several sub-glycoproteomes were obtained by: (i) affinity chromatography on concanavaline A (ConA) and analysis of glycoproteins by MALDI-TOF MS; (ii) multidimensional lectin chromatography (using AIL, PNA, ConA and WGA lectins) and subsequent identification of glycoproteins by MALDI-TOF MS and LC-MS/MS; (iii) boronic acid chromatography followed by identification of glycoproteins by MALDI-TOF MS. Altogether, 127 glycoproteins were identified. Most glycoproteins were found to be putative N-glycoproteins and N-glycopeptides were predicted from MS data using the ProTerNyc bioinformatics software.     

Epidermal growth factor and transforming growth factor-beta1 enhance HK-2 cell migration through a synergistic increase of matrix metalloproteinase and sustained activation of ERK signaling pathway

Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), upregulated in renal diseases, have a combinational effect on epithelial-mesenchymal transformation (EMT) of renal proximal tubular cells. The aim of this study was to examine the mechanism regarding the combinational effect of EGF and TGF-beta1 on cell migration following EMT. The results demonstrated that EGF (10 ng/ml) and TGF-beta1 (3 ng/ml) synergistically increased cell migration, accompanied by an increase in matrix metalloproteinase-9 (MMP-9) gene expression, production and activity. Inhibition of MMP-9 production and activity by an MMP-2/MMP-9-specific inhibitor blocked the synergistic effect of EGF and TGF-beta1 on cell migration. The kinetic profile of extracellular signal-regulated kinase (ERK) signals demonstrated that ERK1/2 activation was rapidly and strongly induced by EGF but delayed and less marked by TGF-beta1 stimulation. In contrast, co-administration of EGF and TGF-beta1 caused an early pronounced and persistent ERK1/2 activation. Inhibition of the ERK1/2 activity by PD98059 abrogated the synergistic effect of EGF and TGF-beta1 on cell migration, MMP-9 production and activity, indicating that EGF and TGF-beta1 converged at the ERK signaling pathway to mediate cell migration. This study demonstrates that EGF and TGF-beta1 synergistically stimulate proximal tubular cell migration through the increased MMP-9 function and enhanced ERK1/2 activation.     

A semi-empirical model for cell kill kinetics during hyperthermia

A three-parameter mathematical model is developed which describes the available data on cell-kill kinetics during hyperthermia. The sub-exponential behaviour of the kinetics suggests that the cell-kill is not a one-step process.     

Automation of cell line development

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 μM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 μM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 μM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process. A. Salmén and K. Lindgren contributed equally to the work.     

Chromosomes and their relationship to nuclear components during the cell cycle in Chinese hamster cells

Summary Chromosomes and their relationship to nuclear components during various phases of the cell cycle were studied with different fixation, embedding, and enzyme techniques. The results showed that interphase chromosomes may have oriented in such a way that a given locus became associated with the nuclear membrane. Some chromosomes also appeared to interact with the nucleolus. The nuclear matrix materials, however, were distributed between the chromosomes and formed a delineating boundary for the chromosomes. These matrix materials, furthermore, formed channel-like structures within the nucleus and towards the cytoplasm through their interaction with nuclear pore complexes. During mitosis, chromosomes were encapsulated with material that appeared to be derived from the matrix, disintegrated residues and fragments of the nuclear envelope, the lamina, and nucleolar material. These chromosome-associated materials seen in mitosis appeared to serve as foci for formation of new nuclear components in subsequent interphase.     

Electron-microscopic studies on the physiological cell loss in the gastric mucosa of the golden hamster

Summary Fine-structural aspects of physiological cell loss in the gastric mucosa of the golden hamster were observed. As the surface mucous cell ascends along the gastric pit, the cell becomes taller and funnel-like in shape. The interfoveolar cell located at the superficial portion of the gastric pit has many lysosomes and a few lipid droplets in the cytoplasm. The nucleus moves toward the upper region of the cytoplasm, while the Golgi apparatus moves downward toward the infranuclear region. After the rupture of the apical plasma membrane takes place, the lateral and basal plasma membranes of this cell remain in spite of loss of the cell contents. Between the basal plasma membrane of the interfoveolar cell and the capillary endothelium is a thick connective tissue layer characterized by densely packed collagen fibrils. The remaining basal and lateral plasma membranes of the ruptured cell and the thick underlying collagenous layer might play a role in protecting the tissue from potential damage induced by the physiological cell loss.This study was supported in part by a grant from the Research Fund of the Ministry of Education, Science and Culture, Japan.     

Analysis of Chinese hamster ovary cell metabolism through a combined computational and experimental approach

Optimization of cell culture processes can benefit from the systematic analysis of experimental data and their organization in mathematical models, which can be used to decipher the effect of individual process variables on multiple outputs of interest. Towards this goal, a kinetic model of cytosolic glucose metabolism coupled with a population-level model of Chinese hamster ovary cells was used to analyse metabolic behavior under batch and fed-batch cell culture conditions. The model was parameterized using experimental data for cell growth dynamics, extracellular and intracellular metabolite profiles. The results highlight significant differences between the two culture conditions in terms of metabolic efficiency and motivate the exploration of lactate as a secondary carbon source. Finally, the application of global sensitivity analysis to the model parameters highlights the need for additional experimental information on cell cycle distribution to complement metabolomic analyses with a view to parameterize kinetic models.     

Beneficial effect of silkworm hemolymph on a CHO cell system: Inhibition of apoptosis and increase of EPO production

To produce erythropoietin (EPO), Chinese hamster ovary (CHO) cells were first cultured in a medium containing FBS (growth medium) and then in a serum-free medium containing sodium butyrate (production medium). Sodium butyrate increases recombinant protein production, but also induces apoptosis, which reduces cell viability and productivity. In a previous study, we found that silkworm hemolymph (SH), an insect serum, inhibits the apoptosis of insect and mammalian cells. To overcome sodium butyrate-induced apoptosis, we added SH to growth medium. This pretreatment with SH inhibited the sodium butyrate-induced apoptosis of CHO cells and consequently increased their longevity and their ability to produce EPO. As a result, the volumetric productivity of EPO was increased five-fold. SH was found to inhibit cytochrome c release from mitochondria into the cytosol, and prevented the activation of caspase-3 and other subsequent caspase reactions.     

Chinese hamster x American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase,NADP-1 and malate dehydrogenase,NAD-1

Summary Chinese hamster x American mink somatic cell hybrids were obtained and examined for chromosome content and expression of mink malate dehydrogenase, NADP (MOD-1; EC 1.1.1.40), malate dehydrogenase, NAD (MOR-1; EC 1.1.1.37), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). All the hybrid clones examined were found to segregate mink chromosomes. A clone panel containing 25 clones was set up. The possibilities and limitations of this panel for mink gene mapping are analysed. Using this panel, it is feasible to rapidly map genes located on chromosomes 1–13 and to provisionally assign genes located on chromosome 14 and the X. Based on the data obtained, the genes for MOD-1 and MOR-1 were firmly assigned to mink chromosomes 1 and 11, respectively, and the genes for G6PD and HPRT were provisionally assigned to the X.     

Extracellular NAD and ATP: Partners in immune cell modulation

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as ‘danger signals–to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response.     

Toxoplasma gondii: Growth in the absence of host cell protein synthesis

Host cell protein synthesis continues when cultured cells are infected by Toxoplasma gondii. In order to determine if this host function is necessary for the parasite we used two independent methods that specifically block cellular protein synthesis. In the first, we infected a temperature-sensitive Chinese hamster ovary cell mutant that has a thermolabile leucyl tRNA synthetase. At the restrictive temperature of 40 C, the mutant cells showed only negligible protein synthesis that was probably mitochondrial. At this temperature, the growth and nucleic acid synthesis of T. gondii proceeded normally and [³H]leucine was specifically incorporated into the parasite as demonstrated by autoradiography. A secpnd method for blocking protein synthesis by the host cell employed treatment of uninfected human fibroblast cells with muconomycin A, an inhibitor of initiation. Repeated washing of monolayer cultures reduced the free muconomycin A to an insignificant level while the cells remained incapable of protein synthesis. T. gondii infected and grew normally in the inhibited cells. Autoradiographic localization of the incorporation of [³H]leucine showed that it was almost exclusively in the intracellular parasites in the cells pretreated with muconomycin A. In the untreated control most of the [³H]leucine was incorporated by the host cell rather than the parasite. We conclude that de novo protein synthesis by the host cell is not required to support the growth of intracellular T. gondii.     

Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.     

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in color-ectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data col-lectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels.