A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.     

Secretome analysis using a hollow fiber culture system for cancer biomarker discovery

Secreted proteins, collectively referred to as the secretome, were suggested as valuable biomarkers in disease diagnosis and prognosis. However, some secreted proteins from cell cultures are difficult to detect because of their intrinsically low abundance; they are frequently masked by the released proteins from lysed cells and the substantial amounts of serum proteins used in culture medium. The hollow fiber culture (HFC) system is a commercially available system composed of small fibers sealed in a cartridge shell; cells grow on the outside of the fiber. Recently, because this system can help cells grow at a high density, it has been developed and applied in a novel analytical platform for cell secretome collection in cancer biomarker discovery. This article focuses on the advantages of the HFC system, including the effectiveness of the system for collection of secretomes, and reviews the process of cell secretome collection by the HFC system and proteomic approaches to discover cancer biomarkers. The HFC system not only provides a high-density three-dimensional (3D) cell culture system to mimic tumor growth conditions in vivo but can also accommodate numerous cells in a small volume, allowing secreted proteins to be accumulated and concentrated. In addition, cell lysis rates can be greatly reduced, decreasing the amount of contamination by abundant cytosolic proteins from lysed cells. Therefore, the HFC system is useful for preparing a wide range of proteins from cell secretomes and provides an effective method for collecting higher amounts of secreted proteins from cancer cells. This article is part of a Special Issue entitled: An Updated Secretome.     

接合转移诱动系统在遗传分析和体内基因操作中的应用

接合转移诱动系统在遗传分析和体内基因操作中的应用赵巍,张成刚,蔺继尚（中国科学院沈阳应用生态研究所,１１００１５）细菌间ＤＮＡ的转移主要有转化、转导、接合和原生质体融合等几种形式。接合是通过供体菌和受体菌完整细胞间的直接接触,而传递大段ＤＮＡ的方法,...     

Needle in a haystack: microdissecting the proteome of a tissue

Summary. Laser-assisted microdissection is a recent technology that enables cells to be harvested from tissue sections. Proteins can be extracted from the dissected cells for molecular analysis. This enables the analysis of proteins in specific cell types in an in vivo system. Although quantities of protein obtained from the dissected material can be small, it is possible to use established methods such as Western Blotting and 2D-PAGE, as well as newer technologies such as SELDI-MS, to analyse the proteins. This review describes the applications and technical considerations for using laser-assisted dissected cells in proteomics research.     

Cytopress: automated slide preparation of cytologic material from suspension

This paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure-fixation. Using cervical cells as a model, results show that a well-defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.     

Selective agents and marker genes for use in transformation of monocotyledonous plants

Even the most successful transformation systems for monocotyledonous plants have a very low efficiency. To overcome this, the selection system is of utmost importance. Established selection systems based on kanamycin resistance are not generally applicable for monocotyledonous plants because monocot cells and tissues are relatively insensitive to kanamycin. Selection can be improved by using other antibiotics or herbicides and their respective resistance genes, which can lead to a substantial reduction in the number of untransformed regenerants. In choosing a selection system the resistance mechanism, either detoxification of the selective agent or modification of the target enzyme, should also be taken into account. Detoxification of the selective agent by expression of a modifying enzyme in transformed cells can enable untrasformed cells to escape. This does not happen when resistance is based on the production of a modified target enzyme. By use of vectors specifically designed for high expression in monocots, the level of resistance in transformed cells and tissues can be enhanced, resulting in an increased efficiency of selection. This paper presents an overview of the various antibiotics and herbicides available and the application of the related resistance genes to improve transformation of monocots.     

Blood money

Foetal blood contains cells that can regenerate all the cellular elements of blood and the immune system (stem cells). This blood can be obtained from the placenta without any harm to mother or baby and is currently used as a successful alternative to bone marrow for paediatric transplantation. Recently, it has been suggested that this blood can be put in storage and used (if needed) for the child that the blood originally came from. This has prompted a commercial interest, leading to both financial and ethical concerns.     

Cooperative Regulation of Cellular Proliferation by Intercellular Diffusion

A theoretical model for the cooperative control of cellular kinetics is investigated. A critical substance A is produced by the cells whose concentration in a given cell determines whether that cell can divide. The substance A can leak out of the cells into the surrounding medium as well as be reabsorbed by the cells. This feature then implies communication between the cells since all concentrations will be functions of the population density. The substance A also has a lifetime, i.e. decays, for example, by denaturation. This system can be described by three coupled nonlinear differential equations which can be solved analytically in certain limiting cases and can, of course, be studied in detail by computer techniques. Our investigations have shown that (a) there is a critical initial cell population density below which cell proliferation will not occur, (b) cell proliferation can be stimulated by supplying substance A to the medium and there is a critical initial concentration in the medium for initiating proliferation when the cell population density is subcritical, and (c) a well-defined induction period prior to exponential growth may exist whose length depends on the system parameters and initial conditions.     

Rat kidney proximal tubule cells in defined medium: The roles of cholera toxin,extracellular calcium and serum in cell growth and expression of γ-glutamyltransferase

Summary A culture system is described in which rat kidney proximal tubule epithelial cells (RPTE) can be prepared with good yield and high viability and grown in culture under serum-free conditions. The cells require EGF, insulin, cholera toxin and either 1% dialyzed serum or a complex of bovine serum albumin with oleic acid (BSA/OA). The cells can be maintained for long periods of time and express several markers for RPTE. The cells have both alkaline phosphatase and γ-glutamyltransferase activity and respond to parathyroid hormone but not vasopressin. The specific activity of γ-glutamyltransferase decreases when the cells begin to grow, but increases when they reach confluence. Extracellular calcium plays a role in the induction of γ-glutamyltransferase in confluent cells. Cells grown in media containing low calcium, i.e. less than 0.4 mM, have reduced specific activity of γ-glutamyltransferase. Extracellular calcium also alters the morphology of the cells in that cells grown in low calcium are single cells or loose clusters suggesting poor cell-cell contact. When the calcium is raised to 1.0 mM, the cells change their shape and organization to adopt the morphology of cells maintained continuously in 1.0 mM calcium. The cells can be passaged onto plastic surfaces which have been coated with collagen but cannot be subcultured on uncoated or serum coated plastic. This culture system will be a useful model for the investigation of renal carcinogenesis and the role of cell proliferation in that process. This work was supported by grants CA48197 and DK38925 from the National Institutes of Health Editor's Statement This paper reports a method for isolation and, uniquely, multipassage culture of rat proximal tubule epithelial cells. The authors use this culture system to explore some aspects of the physiology of these cells, demonstrating the value of the model.     

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.     

Are cultured human myotubes far from home?

Satellite cells can be isolated from skeletal muscle biopsies, activated to proliferating myoblasts and differentiated into multinuclear myotubes in culture. These cell cultures represent a model system for intact human skeletal muscle and can be modulated ex vivo. The advantages of this system are that the most relevant genetic background is available for the investigation of human disease (as opposed to rodent cell cultures), the extracellular environment can be precisely controlled and the cells are not immortalized, thereby offering the possibility of studying innate characteristics of the donor. Limitations in differentiation status (fiber type) of the cells and energy metabolism can be improved by proper treatment, such as electrical pulse stimulation to mimic exercise. This review focuses on the way that human myotubes can be employed as a tool for studying metabolism in skeletal muscles, with special attention to changes in muscle energy metabolism in obesity and type 2 diabetes.     

Transport of L-tyrosine by B16/F10 malignant melanocytes: characterization of the process

The main characteristics of L-tyrosine (L-Tyr) uptake by B16/F10 malignant melanocytes are reported. This amino acid can be taken up by two systems, both of them being saturable. The first one would be system L. This system can be studied in cells preloaded with amino acids that are a good substrate for system L, such as L-methionine or L-tryptophan. The kinetic parameters for L-Tyr uptake by this transport system are Vm = 6.5 pmol L-Tyr/10(3) cells.min and Km around 130 microM. The second system, probably the system ASC, shows lower capacity but higher affinity than the former. This system can be detected only in cells previously depleted of amino acids, showing approximate kinetic values of Vm 0.05 pmol L-Tyr/10(3) cells.min and Km around 5 microM. It is shown that the increase in cell density yields a decrease in the rate of L-Tyr uptake by system L, but this increase does not affect the high affinity system, alpha-MSH does not affect significantly the L-Tyr uptake by both systems. 2-Amino bicyclo-(2,2,1)-heptane-2-carboxylic acid produces a remarkable inhibition of the rate of L-Tyr uptake, but alpha-methylaminoisobutyric acid does not affect the rate of transport of this amino acid. The absence of sodium produces a slight but reliable decrease in the rate of L-Tyr uptake, supporting the involvement of two different transport systems. The ionophores monensin and nigericin enhance the transport by system L, but this effect is suppressed by the presence of ouabain. This finding indicates that the (Na+ -K+)-ATPase is essential for the stimulating action of ionophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential survival as an indicator of potential mutagenicity using repair deficient strains of Saccharomyces cerevisiae and Schizosaccharomyces pombe

A method is presented to screen chemicals for potential mutagenicity on the basis of their ability to cause more killing in cells of repair-deficient yeast than in wild type cells. Two species were chosen in the event that one might be more sensitive to certain chemicals. The strains used were RAD+ and rad6 derivatives of Saccharomyces cerevisiae and RAD+ and rad3 derivatives of Schizosaccharomyces pombe. This report describes the test system and results for 12 known, direct-acting mutagens (i.e., not requiring mammalian metabolic activation). These compounds showed more lethality in one or both of the repair-deficient strains, indicating that they induce damage to DNA which is subject to repair in wild type cells. Advantages of this system include the use of eukaryotic yeast cells which can be manipulated as easily as bacteria, and that exogenous enzymes (S9) can be added for metabolic activation. Growing yeast cells can activate certain promutagens, and preliminary experiments showed positive responses for diethylnitrosamine and 2-acetylaminofluorene without the addition of S9.     

Gene amplification system based on double rolling-circle replication as a model for oncogene-type amplification

Gene amplification contributes to a variety of biological phenomena, including malignant progression and drug resistance. However, details of the molecular mechanisms remain to be determined. Here, we have developed a gene amplification system in yeast and mammalian cells that is based on double rolling-circle replication (DRCR). Cre-lox system is used to efficiently induce DRCR utilizing a recombinational process coupled with replication. This system shows distinctive features seen in amplification of oncogenes and drug-resistance genes: (i) intra- and extrachromosomal amplification, (ii) intensive chromosome rearrangement and (iii) scattered-type amplification resembling those seen in cancer cells. This system can serve as a model for amplification of oncogenes and drug-resistance genes, and improve amplification systems used for making pharmaceutical proteins in mammalian cells.     

New perspectives into bacterial DNA transfer to human cells

The type IV secretion system (T4SS) VirB/D4 of the facultative intracellular pathogen Bartonella henselae is known to translocate bacterial effector proteins into human cells. Two recent reports on DNA transfer into human cells have demonstrated the versatility of this bacterial secretion system for macromolecular substrate transfer. Moreover, these findings have opened the possibility for developing new tools for DNA delivery into specific human cell types. DNA can be introduced into these cells covalently attached to a site-specific integrase with potential target sequences in the human genome. This novel DNA delivery system is discussed in the context of existing methods for genetic modification of human cells.     

Regulatory genes controlling cell fate choice in embryonic and adult neural stem cells

Neural stem cells are the most immature progenitor cells in the nervous system and are defined by their ability to self-renew by symmetric division as well as to give rise to more mature progenitors of all neural lineages by asymmetric division (multipotentiality). The interest in neural stem cells has been growing in the past few years following the demonstration of their presence also in the adult nervous system of several mammals, including humans. This observation implies that the brain, once thought to be entirely post-mitotic, must have at least a limited capacity for self-renewal. This raises the possibility that the adult nervous system may still have the necessary plasticity to undergo repair of inborn defects and acquired injuries, if ways can be found to exploit the potential of neural stem cells (either endogenous or derived from other sources) to replace damaged or defective cells. A full understanding of the molecular mechanisms regulating generation and maintenance of neural stem cells, their choice between different differentiation programmes and their migration properties is essential if these cells are to be used for therapeutic applications. Here, we summarize what is currently known of the genes and the signalling pathways involved in these mechanisms.     

Rapid determination of plasmid-carrying yeast cells by using an imaging sensor system

A novel imaging sensor system for the determination of plasmid carrying yeast cells was developed. The sensor system consisted of an Silicon Intensifier Target (SIT) video camera, a fluorescent microscope, and a personal computer system equipped with an image memory board. This system was based on the fact that the membrane integrity of only plasmid-carrying cells is lost following cell growth in 5-fluoro-orotic acid (5-FOA) containing medium, and consequently these target cell can be stained with fluorescent probes and detected. In this study, plasmid-carrying cells were detected and their fraction determined in a mixture of both plasmid-carring and plasmid-free cells. A good correlation was observed between the values determined by this sensor system and the conventional method in the 30%-80% range, and one assay was possible within 4 h. This sensor system could be used for the monitoring of plasmid-carrying fraction in recombinant yeast cells during cultivation.     

Family tree analysis of a transformed cell line and the transition probability model for the cell cycle

Variation in intermitotic time between individual cells in culture can be ascribed to the occurrence of random transitions in the cell cycle. We have analysed a family tree of mouse neuroblastoma cells, and observed that variation in difference in intermitotic time between sister cells is smaller than between cousin cells, and this difference is again smaller than between second-cousin and unrelated cells. This observation is incompatible with all transition probability models presented so far. We propose a model for the cell cycle with a single random transition, but with the additional assumption that the (two) system parameters may show variability within the population such that the closer cells are in their relation to each other, the closer their values of the system parameters will be. This model describes correctly the behaviour of the family tree of the cell line and in addition is able to explain why differences in intermitotic time between sister cells are exponentially distributed, while intermitotic times themselves are more or less normally distributed. Methods have been described to quantify the various system parameters.     

The immune system as a regulator of thyroid hormone activity

It has been known for decades that the neuroendocrine system can both directly and indirectly influence the developmental and functional activity of the immune system. In contrast, far less is known about the extent to which the immune system collaborates in the regulation of endocrine activity. This is particularly true for immune-endocrine interactions of the hypothalamus-pituitary-thyroid axis. Although thyroid-stimulating hormone (TSH) can be produced by many types of extra-pituitary cells--including T cells, B cells, splenic dendritic cells, bone marrow hematopoietic cells, intestinal epithelial cells, and lymphocytes--the functional significance of those TSH pathways remains elusive and historically has been largely ignored from a research perspective. There is now, however, evidence linking cells of the immune system to the regulation of thyroid hormone activity in normal physiological conditions as well as during times of immunological stress. Although the mechanisms behind this are poorly understood, they appear to reflect a process of local intrathyroidal synthesis of TSH mediated by a population of bone marrow cells that traffic to the thyroid. This hitherto undescribed cell population has the potential to microregulate thyroid hormone secretion leading to critical alterations in metabolic activity independent of pituitary TSH output, and it has expansive implications for understanding mechanisms by which the immune system may act to modulate neuroendocrine function during times of host stress. In this article, the basic underpinnings of the hematopoietic-thyroid connection are described, and a model is presented in which the immune system participates in the regulation of thyroid hormone activity during acute infection.     

Novel selection marker for mammalian cell transfection

The availability of selectable markers suitable for use in mammalian cells has permitted the analysis of the influence of the stable overexpression of single or multiple genes on specific cell properties. This powerful technique has led directly to many fundamental advances in molecular biology and increased our overall understanding of cell growth and regulatory events. Although a variety of selectable markers are currently available, some cell lines continue to be naturally resistant to certain markers, making direct selection difficult or not feasible. Thus, the characterization of additional cell selectable markers continues to be of interest. We have developed a novel selectable marker based on mitomycin C resistance that is suitable for stable transfection of mammalian cells. This system is based on the ability of the mcrA gene, isolatedfrom Streptomyces lavendulae, to confer mitomycin C resistance to both bacterial and mammalian cells by expression of the MCRA protein. Here we demonstrate that mcrA can be used as a selectable gene marker in Chinese hamster ovary cells when cells transfected with the mcrA gene are either pulsed or cultured continuously with mitomycin C This unique selection system may be of use for transfection of cells that are resistant to currently available selectable markers.