Exploring the Full Power of Combining High Throughput RNAi with High Content Readouts: From Target Discovery Screens to Drug Modifier Studies

Sachse, Christoph; Weiss‐Haljiti, Cornelia; Holz, Christian; Regener, Kathrin; Halley, Francoise; Hannus, Michael; Frenzel, Corina; Kluge, Sindy; Hewitson, Mark; Bader, Benjamin; Burd, Amy; Perkins, Louise M.; Szewczak, Alexander A.; Prechtl, Stefan; Merz, Claudia; Rae, Peter M. M.; Mumberg, Dominik; Echeverri, Christophe J.

doi:10.1002/9780470229866.ch6

Cited by 3 publications

(1 citation statement)

References 19 publications

(21 reference statements)

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“…Assessment of apoptosis and mitosis was conducted using a high-throughput approach [ 23 ]based on computer-assisted automated analysis of captured microscopic digital images [ 24 , 25 ], conducted as part of a fee-for-service arrangement with Cenix Bioscience ( http://www.cenix-bioscience.com/ ). General strategies to minimize false discoveries were used as previously described [ 26 ].…”

Section: Methodsmentioning

confidence: 99%

Genes Associated With Prognosis After Surgery For Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro

2011

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BackgroundMesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype.MethodsWe conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1) in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination.ResultsKnockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only) and tumor cells (all three genes). No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene.ConclusionsWe provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma.

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Section: Methodsmentioning

confidence: 99%

Genes Associated With Prognosis After Surgery For Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro

2011

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Accurate Cytotoxicity and Proliferation Determination: Advantages of a High-Throughput Phenotypic Approach Over ATP Luminescence Assays

Hammerstein

Wylie

2016

ASSAY and Drug Development Technologies

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Cell viability and proliferation assays are a fundamental tool in the drug discovery process and are used to evaluate both the antiproliferative potency and toxicity of compounds. Some lead discovery groups generate cell viability data for up to two million compounds per screen, so any method used to assess these parameters needs to deliver not only on data quality but also on throughput and assay cost per well. Most methods used to determine cell viability cannot deliver on all three of these requirements, so compromises have to be made. Here we show the development and implementation of a cost-effective, no-wash phenotypic assay to simultaneously report the number of cells, percentage of live cells, and cell cycle phase distribution as markers of proliferation and viability. We demonstrate that this assay can be applied to high-density plate formats and be imaged and analyzed in 8 min per plate on a laser scanning imaging cytometer. By comparing the drug-responses of several well-characterized anticancer drugs on HeLa cells, we highlight the key differences between the phenotypic assay and a commercial ATP luminescence detection system.

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Advancing Cancer Drug Discovery Towards More Agile Development of Targeted Combination Therapies

Carragher¹,

Unciti‐Broceta

Cameron

2011

Future Med. Chem.

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Current drug-discovery strategies are typically 'target-centric' and are based upon high-throughput screening of large chemical libraries against nominated targets and a selection of lead compounds with optimized 'on-target' potency and selectivity profiles. However, high attrition of targeted agents in clinical development suggest that combinations of targeted agents will be most effective in treating solid tumors if the biological networks that permit cancer cells to subvert monotherapies are identified and retargeted. Conventional drug-discovery and development strategies are suboptimal for the rational design and development of novel drug combinations. In this article, we highlight a series of emerging technologies supporting a less reductionist, more agile, drug-discovery and development approach for the rational design, validation, prioritization and clinical development of novel drug combinations.

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Exploring the Full Power of Combining High Throughput RNAi with High Content Readouts: From Target Discovery Screens to Drug Modifier Studies

Cited by 3 publications

References 19 publications

Genes Associated With Prognosis After Surgery For Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro

Genes Associated With Prognosis After Surgery For Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro

Accurate Cytotoxicity and Proliferation Determination: Advantages of a High-Throughput Phenotypic Approach Over ATP Luminescence Assays

Advancing Cancer Drug Discovery Towards More Agile Development of Targeted Combination Therapies

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