scholarly journals Multiplexing of Pathway-Specific β-Lactamase Reporter Gene Assays by Optical Coding With Qtracker® Nanocrystals

2009 ◽  
Vol 14 (7) ◽  
pp. 845-852 ◽  
Author(s):  
Thomas Machleidt ◽  
Pamela Whitney ◽  
Kun Bi
Keyword(s):  
Quantum Dots ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Reporter Gene ◽  
Reporter Cell ◽  
Necrosis Factor Alpha ◽  
A Cell ◽  
Multiple Signaling ◽  
Optical Encoding ◽  
Reporter Gene Assays

Reporter assays are widely used in research and drug discovery for analysis of signaling pathways in a cell-based format. Traditionally, reporter gene assays are run in a single-parameter mode, interrogating only 1 pathway per sample. To enable more complex assay formats for pathway analysis, the authors developed a multiplexed reporter cell-based assay that combines optical encoding with a β-lactamase reporter gene readout. The optical encoding is achieved by peptide-mediated delivery of quantum dots into reporter cell lines. Using different quantum dots, the authors were able to simultaneously analyze multiple signaling pathways in the same sample using fluorescence microscopy or flow cytometry. They selected 3 β-lactamase reporter cell lines for the analysis of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon gamma (IFN-γ) induced signaling to perform proof-of-principle experiments. The analysis demonstrates that this multiplexed assay allows the reliable detection of ligand-specific activation patterns as well as pathway-specific inhibitors. This method provides a template for the development of novel assay designs that enable the analysis of complex signaling networks involving multiple signaling pathways as well as cell-specific pathways in heterotypic cell models. ( Journal of Biomolecular Screening 2009:845-852)

scholarly journals The Cardiac Glycoside Deslanoside Exerts Anticancer Activity in Prostate Cancer Cells by Modulating Multiple Signaling Pathways

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5809
Author(s):  
Mingcheng Liu ◽  
Qingqing Huang ◽  
Jun A ◽  
Linyue Li ◽  
Xiawei Li ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Anticancer Activity ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Cardiac Glycoside ◽  
Cardiac Glycosides ◽  
Disease Free Survival ◽  
Diverse Range ◽  
Multiple Signaling

Prostate cancer (PCa) is a leading cause of cancer-related deaths among men worldwide, and novel therapies for advanced PCa are urgently needed. Cardiac glycosides represent an attractive group of candidates for anticancer repurposing, but the cardiac glycoside deslanoside has not been tested for potential anticancer activity so far. We found that deslanoside effectively inhibited colony formation in vitro and tumor growth in nude mice of PCa cell lines 22Rv1, PC-3, and DU 145. Such an anticancer activity was mediated by both the cell cycle arrest at G2/M and the induction of apoptosis, as demonstrated by different functional assays and the expression status of regulatory proteins of cell cycle and apoptosis in cultured cells. Moreover, deslanoside suppressed the invasion and migration of PCa cell lines. Genome-wide expression profiling and bioinformatic analyses revealed that 130 genes were either upregulated or downregulated by deslanoside in both 22Rv1 and PC-3 cell lines. These genes enriched multiple cellular processes, such as response to steroid hormones, regulation of lipid metabolism, epithelial cell proliferation and its regulation, and negative regulation of cell migration. They also enriched multiple signaling pathways, such as necroptosis, MAPK, NOD-like receptor, and focal adhesion. Survival analyses of the 130 genes in the TCGA PCa database revealed that 10 of the deslanoside-downregulated genes (ITG2B, CNIH2, FBF1, PABPC1L, MMP11, DUSP9, TMEM121, SOX18, CMPK2, and MAMDC4) inversely correlated, while one deslanoside-upregulated gene (RASD1) positively correlated, with disease-free survival in PCa patients. In addition, one deslanoside-downregulated gene (ENG) inversely correlated, while three upregulated genes (JUN, MXD1, and AQP3) positively correlated with overall survival in PCa patients. Some of the 15 genes have not been implicated in cancer before. These findings provide another candidate for repurposing cardiac glycosides for anticancer drugs. They also suggest that a diverse range of molecular events underlie deslanoside’s anticancer activity in PCa cells.

scholarly journals Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways

2011 ◽  
Vol 392 (11) ◽  
Author(s):  
Larisa Ring ◽  
Iris Peröbner ◽  
Marisa Karow ◽  
Marianne Jochum ◽  
Peter Neth ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
Reporter Gene ◽  
Luciferase Activity ◽  
Chimeric Protein ◽  
Signaling Cascades ◽  
Hek 293 ◽  
Future Studies ◽  
Wnt Proteins ◽  
293 Cells

Abstract WNT/Frizzled receptor (FZD) signaling pathways are pivotal for physiological and pathophysiological processes. In humans, the complexity of WNT/FZD signaling is based on 19 WNTs, 10 FZDs and at least two (co)receptors (LRP5/6) mediating supposably four different signaling cascades. The detailed investigation of the specific function of the different initiating components is primarily hampered by the lack of most WNT proteins in a purified form. Therefore, we constructed and examined a chimeric protein of WNT3a and FZD4 as a suitable approach to overcome this obstacle for future studies of the specificity of other WNT/FZD combinations. Furthermore, we produced four different reporter HEK 293 cell lines to quantify the induced activation of the proposed signaling cascades, the β-catenin-, the NFAT-, the AP-1- and the CRE-regulated pathways. The chimera WNT3aFZD4 efficiently induced β-catenin-mediated luciferase activity. This activity was increased 40-fold compared with basal when LRP6 was stably cotransfected, proving that the chimera WNT3aFZD4 can also interact efficiently with LRP6. Our results demonstrate that the approach of using reporter gene cell lines in combination with WNT/FZD chimeras is efficient to study the β-catenin-mediated pathway and should also allow clarifying the specificity of WNT/FZD combinations in the activation of the other pathways.

scholarly journals A cell-based ribozyme reporter system employing a chromosomally-integrated 5′ exonuclease gene

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Aiyada Aroonsri ◽  
Jindaporn Kongsee ◽  
Jeremy David Gunawan ◽  
Daniel Abidin Aubry ◽  
Philip James Shaw
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Reporter Gene ◽  
Reporter System ◽  
Ribonucleic Acids ◽  
Reporter Activity ◽  
Biochemical Methods ◽  
Domains Of Life ◽  
A Cell ◽  
Reporter Gene Assays

Abstract Background Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA. Results We established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell. Conclusions The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.

scholarly journals ToxTracker Reporter Cell Lines as a Tool for Mechanism-Based (Geno)Toxicity Screening of Nanoparticles—Metals, Oxides and Quantum Dots

Nanomaterials ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 110 ◽  
Author(s):  
Sarah McCarrick ◽  
Francesca Cappellini ◽  
Amanda Kessler ◽  
Nynke Moelijker ◽  
Remco Derr ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Quantum Dots ◽  
Metal Oxide ◽  
Cell Lines ◽  
Protein Unfolding ◽  
Cellular Stress Response ◽  
Toxicity Screening ◽  
Reporter Cell ◽  
Relevant Dose

The increased use of nanoparticles (NPs) requires efficient testing of their potential toxic effects. A promising approach is to use reporter cell lines to quickly assess the activation of cellular stress response pathways. This study aimed to use the ToxTracker reporter cell lines to investigate (geno)toxicity of various metal- or metal oxide NPs and draw general conclusions on NP-induced effects, in combination with our previous findings. The NPs tested in this study (n = 18) also included quantum dots (QDs) in different sizes. The results showed a large variation in cytotoxicity of the NPs tested. Furthermore, whereas many induced oxidative stress only few activated reporters related to DNA damage. NPs of manganese (Mn and Mn3O4) induced the most remarkable ToxTracker response with activation of reporters for oxidative stress, DNA damage, protein unfolding and p53-related stress. The QDs (CdTe) were highly toxic showing clearly size-dependent effects and calculations suggest surface area as the most relevant dose metric. Of all NPs investigated in this and previous studies the following induce the DNA damage reporter; CuO, Co, CoO, CdTe QDs, Mn, Mn3O4, V2O5, and welding NPs. We suggest that these NPs are of particular concern when considering genotoxicity induced by metal- and metal oxide NPs.

scholarly journals Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases

2006 ◽  
Vol 5 (5) ◽  
pp. 1136-1144 ◽  
Author(s):  
Keiran S.M. Smalley ◽  
Nikolas K. Haass ◽  
Patricia A. Brafford ◽  
Mercedes Lioni ◽  
Keith T. Flaherty ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Melanoma Metastases ◽  
Multiple Signaling
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