Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes

BackgroundThe need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds. Combinatorial chemistry could aid in the search for cisplatin analogs if fast, high-throughput assays were available. Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes. We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells.ResultsHeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared. Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity. By contrast, trans-[Pt(NH3)2Cl2], other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression. Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription. In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-[Pt(NH3)2Cl2] or K2[PtCl4], inhibited β-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2.ConclusionsThe EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription. Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery

Development of a robust GABA B calcium signaling cell line using Β-lactamase technology and sorting

The GABA B receptor is a member of the “family 3” G protein coupled receptors. The GABA B receptors modulate activity inwardly rectifying potassium channels and high voltage activated calcium channels. The GABA B receptors require heterodimerization between two subunits, GABA B1 and GABA B2 , for functional expression. A robust functional calcium cell line was developed that contained both the human truncated GABA B(1b) and human truncated GABA B(2) receptors. The cell line was analyzed and sorted using Β-lactamase as a reporter. Single cell clones were sorted and isolated using flow cytometry based on high Β-lactamase expression. The single cell clones were further tested in a 384-well calcium mobilization assay using the Fluo-4 AM calcium indicator on the fluorescent imaging plate reader system (FLIPR). Twenty-seven clones were grown up from single cell collections and 10 clones demonstrated a high response to GABA stimulation. The 10 clones were re-evaluated based on agonist dose response and EC 50 . Clone-16 was identified and utilized in high throughput screening (HTS) assay development. Using sorting and Β-lactamase as a reporter, we were able to develop a robust, functional cell-based, GABA B , calcium mobilization assay. The cell line described here can be used for high throughput FLIPR screening and also to compare and rank the potency and selectivity of agonists, antagonists and potentiators of the GABA B receptor. © 2008 International Society for Advancement of CytometryPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60462/1/20591_ftp.pd

Relationship between shear energy input and sedimentation properties of exopolysaccharide-producing StreptococcusStreptococcus thermophilusthermophilus strains

separation of the bacteria cells. This separation is most commonly carried out with disc stack separators and needs to be adjusted to the respective strain to obtain a high cell recovery rate. Exopolysaccharides (EPS) produced by several starter cultures, however, have a large negative impact on the separation properties of the cells. These EPS can be divided into cell-bound capsular EPS or free EPS that are released into the surrounding fermentation medium. To improve the separation step, shear forces were applied after fermentation with a gear ring disperser to simulate the impact of a homogenizer and the influence on the separation properties of six Streptococcus thermophilus strains was examined. In case of capsular EPS, the sedimentation velocity of the bacteria increased due to shearing off the capsular EPS layer. Shearing media with free EPS resulted in a viscosity decrease and, hence, in a higher sedimentation velocity, as was determined using a disc centrifuge and a LUMiSizer. Sediment compression as measured with the LUMiSizer was also affected by the shearing step. The results of this study suggest that a defined shear treatment of EPS producing bacterial starter cultures leads to improved separation properties and, hence, higher bacteria yields. We assume that both EPS types affect separation efficiency of the bacteria cells, free EPS because of increased media viscosity and capsular EPS because they act like a friction pad

PI controller tuning for load disturbance rejection using constrained optimization

© 2016, Springer-Verlag Berlin Heidelberg. In this paper, a simple and effective PI controller tuning method is presented. To take both performance requirements and robustness issues into consideration, the design technique is based on optimization of load disturbance rejection with a constraint either on the gain margin or phase margin. In addition, a simplified form of the resulting tuning formulae is obtained for first order plus dead time models. To demonstrate the ability of the proposed tuning technique in dealing with a wide range of plants, simulation results for several examples, including integrating, non-minimum phase and long dead time models, are provided

Articulatory feature recognition using dynamic Bayesian networks.

We describe a dynamic Bayesian network for articulatory feature recognition. The model is intended to be a component of a speech recognizer that avoids the problems of conventional ``beads-on-a-string'' phoneme-based models. We demonstrate that the model gives superior recognition of articulatory features from the speech signal compared with a state of- the art neural network system. We also introduce a training algorithm that offers two major advances: it does not require time-aligned feature labels and it allows the model to learn a set of asynchronous feature changes in a data-driven manner

A destabilized bacterial luciferase for dynamic gene expression studies

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression

Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

BACKGROUND: The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. METHODS: AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. RESULTS: Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a β-lactamase reporter gene, a 10–12 fold shift or 2.5–3 fold shift toward greater potency in the IC(50 )was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. CONCLUSION: EGFR pathway components were qualified as targets for inhibition of AP-1 activation using RNAi and small molecule inhibitors. The combination of these two targeted agents was shown to increase the efficacy of EGFR and MEK-1 kinase inhibitors, leading to possible implications for overcoming or preventing drug resistance, lowering effective drug doses, and providing new strategies for interrogating cellular signalling pathways

Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform