HPLC-ED measurement of endogenous catecholamines in human immune cells and hematopoietic cell lines

A rapid and simple HPLC-ED method is described to identify and measure catecholamines (CTs) and their major metabolites in immune cells. Using this method, intracellular CTs were quantified in human peripheral blood mononuclear cells (PBMCs), T and B lymphocytes, monocytes and granulocytes. Immune cell subsets were separated by density gradient centrifugation and immunomagnetic cell sorting. CTs were also found in the human hematopoietic cell lines NALM-6 (pre-B) and (in smaller amounts) in Jurkat (T lymphoblastoid) and U937 (promonocytic). In cultured PBMCs, intracellular CTs were reduced by both the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine and the chromaffin granule depletant reserpine. In NALM-6 cells, both alpha-methyl-p-tyrosine and the dopamine-beta-hydroxylase inhibitor disulfiram reduced intracellular CTs, supporting the presence of active synthetic pathways in these cells. Since sympathoadrenergic mechanisms play a key role in the interactions between the immune system and the nervous system, these findings may be relevant for a better understanding of the neuro-immune network.     

Generation and application of signaling pathway reporter lines in zebrafish

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.     

Generation and characterization of human insulin-releasing cell lines

Background  

The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines.     

TRACE generates fluorescent human reporter cell lines to characterize epigenetic pathways

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Distinct differentiation characteristics of individual human embryonic stem cell lines

Background  

Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic.     

Anti-proliferative action of endogenous dehydroepiandrosterone metabolites on human cancer cell lines

Dehydroepiandrosterone (DHEA) is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal tract, and it is known to have chemopreventive and anti-proliferative actions on tumors. These effects are considered to be induced by the inhibition of glucose-6-phosphate dehydrogenase (G6PD) and/or HMG-CoA reductase (HMGR) activities. The present study was undertaken to investigate whether endogenous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated DHEA derivatives, androsterone, epiandrosterone, and etiocholanolone, have anti-proliferative effects on cancer cells and to clarify which enzyme, G6PD or HMGR, is responsible for growth inhibition. Growth of Hep G2, Caco-2, and HT-29 cells, evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays, was time- and dose-dependently inhibited by addition of all DHEA-related steroids we tested. In particular, the growth inhibition due to etiocholanolone was considerably greater than that caused by DHEA in all cell lines. The suppression of growth of the incubated steroids was not correlated with the inhibition of G6PD (r=-0.031, n=9, NS) or HMGR (r=0.219, n=9, NS) activities. The addition of deoxyribonucleosides or mevalonolactone to the medium did not overcome the inhibition of growth induced by DHEA or etiocholanolone, while growth suppression by DHEA was partially prevented by the addition of ribonucleosides. These results demonstrate that endogenous DHEA metabolites also have an anti-proliferative action that is not induced by inhibiting G6PD or HMGR activity alone. These non-androgenic DHEA metabolites may serve as chemopreventive or anti-proliferative therapies.     

Generation of a fluorescently labeled endogenous protein library in living human cells

We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100-200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3' rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format.     

Insect cell lines as tools for studying ecdysteroid action

Summary

Recent research concerning ecdysteroid-responsive and ecdysteroid-producing cell lines is reviewed. The advantages and limitations of cell lines of defined and undefined origin are considered with regard to their suitability for studies on molecular, physiological, morphological and developmental aspects of ecdysteroid action. The considerable potential for future studies involving insect cell lines is indicated.     

Paneling human thyroid cancer cell lines for candidate proteins for targeted anti-angiogenic therapy

Tumor angiogenesis is believed to result from an imbalance of pro- and anti-angiogenic factors, some of which are candidates for targeted therapy. Such therapy has raised hopes for patients with undifferentiated thyroid carcinomas, who are facing a grave prognosis with a survival of only months. In this study, in vivo growth of xenografted human thyroid carcinomas unexpectedly responded quite differently to neutralizing anti-vascular endothelial growth factor (VEGF) antibody. In particular, lasting inhibition as well as accelerated growth occurred after treatment. Consequently, a panel of anti-angiogenic factors was addressed in a representative sample of thyroid carcinoma lines. VEGF, fibroblast growth factor (FGF-2), and endostatin were demonstrated by Western blotting and EIA, whereas PDGF-A, PDGF-B, and IL-6 were negative. Quantification of VEGF, FGF-2, and endostatin revealed a wide range of concentrations from 500 to 4,200 pg/ml VEGF, 5 to 60 pg/ml FGF-2, and 50 to 300 pg/ml endostatin, not related to a particular histologic thyroid carcinoma background. Angiostatin (kringles 1-3) was detected in all, but one of the cell lines. Finally, aaATIII was confirmed in FTC133 cells. These data highlight the complex regulation of angiogenesis in thyroid carcinoma cell lines and suggest that the array of angiogenic factors differs markedly between individual cell lines. For the first time, angiostatin, endostatin, and possibly also aaATIII are identified as novel candidate regulators of angiogenesis in thyroid carcinoma cells.     

Generation of Nppa‐tagBFP reporter knock‐in mouse line for studying cardiac chamber specification

Nppa is a cardiac hormone which plays critical roles in regulating salt–water balance. Its expression is restricted to the atria of the healthy post‐natal heart. During heart development, spatio‐temporal expression of Nppa is dynamically changed within the heart and becomes restricted to the atria upon birth. In contrast to its atrial specific expression after birth, Nppa is re‐expressed in the adult ventricles in response to cardiac hypertrophy. To study cardiac chamber specification during development and pathological cardiac remodeling during heart disease, we generated a novel Nppa reporter mouse line by knocking‐in a tagBFP reporter cassette into 3′‐UTR of the Nppa gene without disrupting the endogenous gene. Our results demonstrated dynamic tagBFP expression in the developing heart, recapitulating the spatiotemporal expression pattern of endogenous Nppa. We also found that Nppa‐tagBFP is induced in the ventricle during pathological remodeling. Taken together, Nppa‐tagBFP reporter knock‐in mouse model described in this article will serve as a valuable tool to study cardiac chamber specification during development as well as pathological cardiac remodeling.     

A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors

Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively.     

Generation of long-term human cytolytic cell lines with persistent natural killer activity

Human cell lines maintained by in vitro stimulation with the HLA-A, B-negative, DR-positive, Epstein Barr virus-transformed, lymphoblastoid cell line Daudi in the presence of conditioned medium demonstrated significant NK activity for over 6 wk in continuous culture. These cells lyse K562 and a broad panel of lymphoblastoid cell lines but do not lyse normal peripheral blood lymphocytes or pokeweed mitogen blasts. They possess the sheep red blood cell receptor but lack other T cell markers (Lyt-3+, OKT3-). Natural killer activity correlated with the presence of a Mac 1-positive subpopulation of cells present in these long-term lines.