Notes: CellTiter-Glo® Cell Viability Assay was used to assess the vulnerability of myc-driven cancers to inhibitors of the NAMPT. Sensitivity was correlated to the level of myc expression. Apoptosis was assessed with the Caspase-Glo® 3/7 Assay. The NAD/NADH-Glo® Assay was used to provide a quantitative measure of NAD+ in the cells under study. (4835)

Notes: The authors demonstrate that the X-linked inhibitor of apoptosis (XIAP) drives anti-EGFR and anti-HER antibody resistance in inflammatory breast cancer both through its effect on caspase and suppression of ROS accumulation. They use the CytoTox ONE Homogeneous Membrane Integrity Assay to assess cytotoxicity in wild type SUM149 and SUM190 cells, and  rSUM149 and rSUM190 cells (cell lines that have acquired resistance to treatment agents) upon treatment with cytotoxic agents. The Caspase-Glo® 3/7 Assay was used to assess apoptosis in wild type cells, rSUM149 and in cells over expressing XIAP (treated and untreated). (4726)

Notes: The authors of this study sought to determine whether cucurbitacin B has antitumor activity against the ovarian cancer cell line A2870 and whether it can sensitize the cisplatin-resistant cell line A2780CP to treatment. They compared caspase activity in A2780CP cells either preincubated with cucurbitacin B before treatment with cisplatin or cells not pretreated using the Caspase-Glo® 3/7 Caspase Assay. Oxidized and total glutathione were measured from both cell lines (before and after cisplatin exposure, with and without preincubation with cucurbitacin B) using the GSH/GSSH-Glo™ Glo Assay. The level of reactive oxygen species in cell lines was also measured by detecting ROS converted to H₂O₂ using the ROS-Glo™ Assay. Cell Viabilty of 3D spheroids formed from the A2780CP cell line was assessed using the CellTiter-Glo® 3D Cell Viability Assay. (4581)

Notes: The authors describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. They monitored cell health for 72 hours from the same test samples, distinguished differential cell growth, and investigated drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements enabled them to detect cell death immediately (>75% signal decrease within 15 minutes of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects.They then screened an oncology compound library (Z′ = 0.7) and identified compounds with varying activity at different time points. (4590)

Notes: Low-temperature atmospheric pressure plasmas (LTP) were examined for the mechanism of toxicity on cultured immortalized and primary prostate cancer cells. LTP induces an increase in H₂O₂ in the culture media as judged by the ROS-Glo™ H₂O₂ Assay leading to cell death as measured with the CellTox™ Green Cytotoxicity Assay. CellTox™ Green was quantified with a plate reader and verified by fluorescent microscopy. The cells experienced no caspase-3/7 activation as judged through use of the Caspase-Glo® 3/7 Assay, leading to the conclusion of necrotic cell death. (4711)

Notes: A phenotypic screen of a 25,000 compound library for inhibitors of U2OS osteosarcoma cell line growth identified two compounds that showed activity of osteosarcomas and not HepG2 or primary human osteoblasts.  Cell Viability in the primary and secondary screens was monitored with the CellTiter-Blue® Cell Viability Assay.  Subsequent studies identified the mechanism of cell death as apoptosis as judged by the Caspase-Glo® 3/7 Assay and cytotoxicity measured with the CellTox™ Green Cytotoxicity Assay.
(4649)

Notes: 3T3-L1 preadipocytes were treated with various levels of reversine and monitored for induction of apoptosis with the Caspase-Glo® 3/7 Assay with data collected on a GloMax® Instrument. Changes in gene expression of three targets upon reversine treatment were examined through total RNA isolation with the ReliaPrep™ RNA Cell Miniprep System, reverse transcription with the ImProm-II™ Reverse Transcription System followed by dye-based qPCR analysis. (4596)

Notes: Caspase-Glo® 3/7 and Caspase-Glo® 8 Assays were used to assess activation of apoptosis pathways in MC3T3-E1 cells (clonal mouse), U-2 OS human osteoscarcoma cell line and CCL-51 cells (mouse mammary gland tumor cells). Although ibandronate reduced cell proliferation in all cell lines, its effect on activation of caspases was different in neoplastic versus non-neoplastic cells. Caspase-8 and caspase-3/7 activities were reduced in MC3T3-E1 cells after 72 hours treatment with ibandronate. In two tumor cell lines assayed, opposite results were seen: caspase-8 and caspase-3/7 activities increased in U-2 OS cells and in CCL-51 cells. Luminescence was detected using a GloMax® 96 Microplate Luminometer. (Figure 1 in the paper)

To analyze FAS promoter methylation levels, fragments of the targeted promoter regions were generated by digestion of genomic DNA using CpG methylation insensitive restriction enzymes MboII and PstI from cells cultured in the presence of ibandronate for varying lengths of time. The authors demonstrated that FAS promoter methylation is altered in tumor cells in the presence of ibandronate. (4253)

Notes: The E3 ubiquitin ligase murine double minute 2 (MDM2) regulates transcriptional activity and expression levels of the p53 tumor suppressor protein. In this article, the authors show that the cell fate determinant NUMB interacts with MDM2, disrupting the interaction of MDM2 and p53 and preventing the ubiquitination of p53. To study protein-protein interactions between NUMB and MDM2, the authors used the pFN2A (GST) Flexi® Vector to express full-length and partial NUMB sequences as GST fusion proteins for protein pull-down experiments. To examine their hypothesis that the interaction between NUMB and MDM2 increases p53 levels and thus apoptosis in cells, they treated ZR75 human breast carcinoma cells and A375 human melanoma cells with NUMB-derived peptides and small molecule ligands that block the interaction between NUMB and MDM2. These treatments resulted in increased levels of p53 protein, Annexin V staining as well as caspase-3/7 activity, as determined using the Caspase-Glo® 3/7 Assay System. (4351)

Notes: These authors investigated the ability of Phyllanthus plant extracts to affect the metastatic activity of human lung (A549) and breast (MCF-7) cancer cell lines. They initially used CellTiter 96® AQ_ueous Non-Radioactive Cell Proliferation Assay and the GloMax®-Multi Detection System absorbance module to determine cytotoxicity of various Phyllanthus extracts. After determining the effective dose, the authors investigated the ability of these plant compounds to inhibit/reduce metastatic activity. They then evaluated the mechanism of cell death in treated cells using the Caspase-Glo® 3/7 Assay and the Glomax® Multi Detection System luminescence module to measure caspase activity, and the CytoTox-ONE™ Homogeneous Membrane Integrity Assay to measure LDH activity. (4196)

Notes: In this study, caspase-3/7 activity in HUVECs was measured using the Caspase-Glo^® 3/7 Assay. Luminescence was measured with the GloMax^®-96 Microplate Luminometer. (4207)

Notes: The authors of this study set out to describe the mechanism of cell death through which TNF-like weak inducer of apoptosis (TWEAK) exerts its apoptotic effect on certain cancer cells. The used the CellTiter-Glo® Cell Viability Assay and the Caspase-Glo® 3/7 Assay to investigate cell viability and mechanism of cell death in HSC3 cells treated with TWEAK. They looked at caspase-8 activity in cells treated with TWEAK in the presence or absence of a caspase-8 inhibitor using the Caspase-Glo® 8 Assay. They showed that TWEAK induces caspase-dependent apoptosis in these cells. (4170)

Notes: Dysregulation of the NF-kB pathway has been associated with the formation of a wide variety of tumors and other cancers, as well as diseases, including chronic inflammation and immunodeficiency. Because of the association of constitutive NF-kB regulation and tumors, inhibition of the NF-kB pathway by small molecule antagonists was thought to be a means of reversing or halting the growth and spread of tumors. The authors screened compounds from a database (the NCGC Pharmaceutical Collection or NPC) of small molecule compounds: 52% of the compounds have been approved for human or animal use by the FDA, 22% were drugs approved for use in Europe, and another 25% either drugs approved in other countries or compounds that have been tested in clinical trials. The database served as a source from which to rapidly and efficiently identify already approved drugs that inhibited NF-kB. They used a quantitative high-throughput screening format. To identify small molecules that could inhibit the NF-kB pathway, the compounds were initially screened using a cell-based NF-kB lactamase reporter gene assay, with TNFalpha and MG132 as positive controls. (TNFalpha induced NF-kB coupled beta-lactamase activity, while MG132 blocked TNFalpha induction NF-kB-coupled beta-lactamase activity.) After several rounds of screening, 20 compounds were further studied for their NF-kB inhibition, with NF-kB luc2P HEK293 cells. After a concordance rate of 95% between the luciferase and beta-lactamase tests, compounds were additionally examined for their ability to affect caspase 3/7 activity, for the ability to disrupt the electrochemical gradient across the mitochondrial membrane in relation to cellular apoptosis, as well as tests of the inhibitors on cancer cell viability and affects on LDH release, an indicator of cell necrosis.
(4049)

Notes: The authors review the use of in vitro cytotoxicity testing in drug discovery to characterize the toxic potential of new chemical entities (nce) at the earliest stages of profiling. DOI: 10.2174/1875397300903010033 (4000)

Notes: The authors describe a multiplexed in vitro assay to detect nephrotoxicity and gain information about mechanism of cell death in HK-2 (human kidney-2) cells. The multiplexed assay involved an LDH assay to detect necrosis, a caspase-3/7 assay to detect apoptosis, a reazurin assay to assess metabolic state, and a DNA dye staining assay to monitor nuclear morphology. (4002)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: These authors showed that the ETS transcription factor Erg interacts with the VE-cadherin promoter region and regulates endothelial apoptosis through this interaction. They demonstrated that inhibition of Erg by siRNA resulted in decreased VE-cadherin mRNA and protein levels, and showed that Erg interacts with the VE-cadherin promoter using a CHIP assay. To show the functional relevance of this interaction, HeLa cells were transfected with a pGL4 Vector containing the VE-cadherin promoter region and an expression vector containing Erg2 cDNA. In this reporter assay, Erg overexpression resulted in ~1.8 fold transactivation of VE-cadherin promoter activity, as measured using the Dual-Luciferase® Reporter Assay System. Inhibition of Erg in human umbilical vein endothelial cells also resulted in a loss of viability and an increase in activation of caspase 3 and caspase 7. The authors showed that apoptosis could be significantly decreased by overexpression of VE-cadherin, indicating that Erg regulates survival partially via its interaction with VE-cadherin. The Caspase-Glo® 3/7 Assay was used to measure caspase activity in these experiments. (3872)

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

Notes: This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms. (3618)

Notes: The authors sought to determine the mechanism by which first-trimester trophoblasts resist FAS ligand-induced apoptosis but remain sensitive to TNFα-mediated apoptosis. First trimester trophoblasts express XAF1 [X-linked inhibitor of apoptosis (XIAP)-associated factor 1], which may be involved in regulating their response to proapoptotic signals. The authors created HaloTag™-XAF1 fusion constructs and transiently transfected the first trimester trophoblast cell line (3A). Cells were labeled with the HaloTag™ TMR ligand, and XAF1 was shown to localize to the cytoplasm. 3A cells transiently transfected with the fusion construct were also separated into cytoplasmic and mitochondrial fractions. The fractions were labeled with HaloTag™ TMR ligand. Expression of the fusion peaked at 48 hours after transfection in both mitochondrial and cytoplasmic fractions. TNFα-treatment of 3A cells induced translocation of endogenous XAF1 to the mitochondria. The authors used the Caspase-Glo® Assays to demonstrate activation of caspase-3 and caspase-9 in response to expression of XAF-1. They also show that caspase-3 activation and XIAP cleavage correlate with translocation of endogenous XAF1 to mitochondria. Viability of 3A and primary trophoblasts over expressing XAF1 was evaluated using the CellTiter® 96 AQueous One-Solution Assay. (3760)

Notes: To examine the role of cyclin-dependent kinase 11 in Fas-induced apoptosis, the authors studied caspase-3 activation in A375 (human melanoma) cells after inducing cell death via Fas. Twenty-four hours after seeding 1 ×10⁴ cells per well in a 96-well plate, 0.5µg/ml anti-Fas antibody was added and the cells incubated 3–72 hours. Activation was determined by adding 100µl Caspase-Glo® 3/7 reagent to each well, incubating for 1 hour and measuring luminescence using a Sirius Luminometer (Berthold Detection System). (3315)

Notes: Wildtype or p53-knockout mouse embryo fibroblasts (MEFs) were infected with 2 MOI A/Puerto Rico/8/34 (PR8) influenza virus. After washing, DMEM containing 1% FBS was added and the cells were grown for 8 or 24 hours before adding the Caspase-Glo® 3/7 Reagent. After 1 hour, luminescence was measured and the fold increase was determined by comparing the RLUs of infected with mock-infected cells. In addition, the Luciferase Assay System was used to assess the level of p53 induction after PR8 infection. Either human primary lung or A549 cells were transfected with a p53-responsive firefly luciferase vector, subsequently infected with 2 MOI PR8 and assessed for reporter enzyme activity 4–24 hours post-infection. (3318)

Notes: The Caspase-Glo™ Assay was used to monitor apoptosis in bovine neutrophils. In these experiments, the researchers slightly modified the Caspase-Glo™ add-and-read procedure. One million neutorphils were treated with a combination of TNF-α and/or gliotoxin for 6 hours before being lysed in a solution of PBS supplemented with protease inhibitors and 1% saponin. The lysates were cleared, stored at -80°C, and then diluted before being analyzed with the Caspase-Glo™ Assay System. Data was presented as the relative light units for each sample. (3305)

Notes: Differences in the activation of apoptotic pathways by the TRAIL death receptors DR4 and DR5 were investigated. An siRNA screen was performed on HCT15 cells in 384-well plates to find genes that influence DR4- or DR5-mediated apoptosis differently. After induction of apoptosis using anti-DR4 or DR5 cross-linked antibodies, cell viability was assessed using the CellTiter-Glo® Reagent. It was found that the Signal Recognition Particle (SRP) plays a major role in DR4- but not DR5-mediated apoptosis. To further investigate this difference, stable HeLa cell lines were generated that expressed short hairpin RNA (shRNA) directed against SRP. The effects of various inducers of apoptosis were tested in these cell lines. Caspase activation was measured using the Caspase-Glo® 3/7 reagent. (3191)

Notes: In this paper, researchers examined the effect of activating mammalian homologues of the Drosophila Toll receptor gene (TDR) on apoptosis in the trophoblast cell lines HTR8 and 3A. Bacterial peptidoglycan (PDG) was used to indirectly activate caspase-3/7, -8 and -9 through TDRs. Caspase activity was measured using the Capase-Glo™ 3/7, 8 and 9 Assays. Caspase-3/7, -8 and -9 activities decreased in both the HTR8 and 3A cell lines transiently transfected with a Fas Associated Death Domain (FADD-DN). Data were expressed as relative light units and were normalized to protein levels in lysates. (3171)