A high throughput screening system of coils for ELF magnetic fields experiments: proof of concept on the proliferation of cancer cell lines

407 Background: Chemotherapy for urothelial cancer (UC) is less effective in patients with large volume metastatic disease, likely due to acquired driver mutations. Driver mutation may lead to differences in sensitivity between primary cell lines and metastatic clones. Quantitative high throughput screening (qHTS) assesses large drug libraries to evaluate in vitro effectiveness. We utilized this modality to compare effectiveness of cell inhibition in two bladder cancer cell lines with their metastatic lines. Methods: We screened T24 and UMUC-3 bladder cancer cell lines against 1,912 oncology-focused drugs using a 48 hr cell proliferation assay with an ATP-based readout (CellTiterGlo), and determined the activity and potency of the compounds in a dose response manner. We compared the results of their activity with known metastatic lines for reach of these primary lines: T24T, SLT3 and FL3 for T24 and LUL2 UMUC3 and UMUC3-luc (LUC3). We identified candidate drugs based on two parameters: 1) more than 70% inhibition at 48 hours and 2) a curve class of -1.1 or -1.2 indicating curve class with good fit (r2 > 0.9). We used a custom capture next generation DNA sequencing chip for exonic regions of 229 known cancer related genes to compare primary and metastatic tumor cell lines. Results: The qHTS compounds, are demonstrated in figure 1a for T24 cell lines and figure 1b for UMUC cell lines. Amongst 1912 drugs tested, only 141 (7.4%) met inclusion criteria for T24. However, only 79 of these drugs (56%) met criteria for all cell lines derived from T24. 160 (8.4%) compounds were found to be active in the UMUC line. Only 32 (20%) of the UMUC3 detected compounds remained active in all UMUC3 derived cell lines. Genomic comparison between cell lines identified several mutations which were found only in the derived cell lines (RAC1 and CDH1 in UMUC3) and (AR, CDK12, GATA1, GUCY1A2 in T24). Conclusions: qHTS of a library of potential therapeutic interventions can produce a list of targets and therapies for bladder cancer. This data reinforces the differences between primary and metastatic tumors as compounds identified active in the primary cell lines were inactive in known metastatic cancer cell lines .

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Identification of a chemical modulator of EZH2-mediated silencing by cell-based high-throughput screening assay

The Journal of Biochemistry ◽

10.1093/jb/mvz007 ◽

2019 ◽

Vol 166 (1) ◽

pp. 41-50 ◽

Cited By ~ 3

Author(s):

Akihiro Murashima ◽

Keiko Shinjo ◽

Keisuke Katsushima ◽

Tetsuo Onuki ◽

Yasumitsu Kondoh ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

High Throughput Screening ◽

Target Genes ◽

Cancer Cell Lines ◽

Methyltransferase Activity ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Catalytic Inhibitor

Abstract Dysregulation of enhancer of zeste homologue 2 (EZH2), a methyltransferase component of polycomb repressive complex 2, is found in many types of cancers especially those that are highly progressive and aggressive. Specific catalytic inhibitors of EZH2 have high anti-tumour activity, particularly in lymphomas with EZH2 activating mutations. However, the clinical benefits of EZH2 catalytic inhibitors in tumours overexpressing EZH2 are still limited. Here, we identified NPD13668, a novel modulator of EZH2-mediated gene silencing, from 329,049 small chemical compounds using a cell-based high-throughput screening assay. NPD13668 reactivated the expression of silenced H3K27me3 target genes together with depletion of the H3K27me3 modification. In addition, NPD13668 repressed the cell growth of prostate cancer cell lines (PC3 and LNCaP) and ovarian cancer cell lines (SKOV3 and NIH-OVCAR3). NPD13668 partially inhibited the methyltransferase activity of EZH2 in vitro. Genome-wide expression analysis revealed that after NPD13668 treatment, about half of the upregulated genes overlapped with genes upregulated after treatment with GSK126, well-known EZH2 catalytic inhibitor, indicating that NPD13668 is a potential modulator of EZH2 methyltransferase activity. Our data demonstrated that targeting the pharmacological inhibition of EZH2 activity by NPD13668 might be a novel cancer treatment.

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Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219873068 ◽

2019 ◽

Vol 25 (1) ◽

pp. 9-20 ◽

Cited By ~ 2

Author(s):

Olivia W. Lee ◽

Shelley Austin ◽

Madison Gamma ◽

Dorian M. Cheff ◽

Tobie D. Lee ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

High Throughput Screening ◽

Drug Targets ◽

Large Scale ◽

Early Stage ◽

Cancer Cell Line ◽

Hek 293 ◽

Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

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The use of human cancer cell lines as a primary screening system for antineoplastic compounds

European Journal of Cancer and Clinical Oncology ◽

10.1016/0277-5379(84)90169-x ◽

1984 ◽

Vol 20 (7) ◽

pp. 947-954 ◽

Cited By ~ 26

Author(s):

Graeme J. Finlay ◽

Bruce C. Baguley

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Human Cancer ◽

Cancer Cell Lines ◽

Screening System ◽

Human Cancer Cell ◽

Human Cancer Cell Lines ◽

Primary Screening

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High-Throughput RNA Interference Screening Identifies Synthetic Lethality Between Oncogenic KRAS Dependency and Suppression of STK33

Blood ◽

10.1182/blood.v112.11.3806.3806 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3806-3806

Author(s):

Claudia Scholl ◽

Stefan Frohling ◽

Ian F. Dunn ◽

David A. Barbie ◽

Anna C. Schinzel ◽

...

Keyword(s):

Rna Interference ◽

Protein Kinase ◽

Cell Viability ◽

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

Synthetic Lethality ◽

Human Cancer ◽

Cancer Cell Lines ◽

Dependent Manner

Abstract Activating RAS mutations are among the most common pathogenetic events in a broad spectrum of hematologic malignancies and epithelial tumors. However, oncogenic RAS has thus far not proven to be a tractable target for therapeutic intervention. An alternative to direct targeting of known oncogenes is to perform “synthetic lethality” screens to identify genes that are selectively required for cell viability in the context of specific cancer-causing mutations. Using this approach, we have discovered a synthetic lethal interaction between mutant KRAS, the most frequently mutated oncogene in human cancer, and inactivation of the gene encoding the STK33 serine/threonine protein kinase. To identify genes that are essential for cell viability in the context of mutant KRAS, we performed high-throughput loss-of-function RNA interference (RNAi) screens in eight human cancer cell lines (mutant KRAS, n=4; wildtype KRAS, n=4), representing seven different tumor types (acute myeloid leukemia, multiple myeloma, colon cancer, breast cancer, ovarian cancer, prostate cancer, glioblastoma), as well as normal human fibroblasts and mammary epithelial cells. We screened each cell line with a subset of the short hairpin RNA (shRNA) library developed by the RNAi Consortium (http://www.broad.mit.edu/genome_bio/trc/rnai.html) that consists of 5,024 individual shRNA constructs targeting 1,011 human genes, including the majority of known and putative protein kinase and phosphatase genes and a selection of known cancer-related genes. In these cell lines, suppression of STK33 preferentially inhibited the viability and proliferation of cells that were dependent on mutant KRAS. The differential requirement for STK33 based on oncogenic KRAS dependency was confirmed in 16 additional cell lines using in vitro transformation assays and human tumor xenograft models. Biochemical analyses support the hypothesis that STK33 promotes cell growth and survival in a kinase activity-dependent manner by regulating the activity of S6K1 as well as BAD-induced apoptosis selectively in mutant KRAS-dependent cells. Notably, molecular genetic characterization of cancer cell lines and analysis of patient-derived genomic data sets indicate that STK33 is not frequently mutated or overexpressed in human tumors. These observations identify STK33 as a potential target for the treatment of mutant KRAS-driven cancers that may have a broad therapeutic index in normal versus malignant cells, and illustrate the potential of RNAi for discovering critical functional dependencies created by oncogenic mutations that cannot be identified using other genomic technologies.

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Selection of lung cancer-associated scFv antibodies from cultured cells

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e22137 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e22137-e22137

Author(s):

P. P. Massion ◽

T. V. Pedchenko ◽

D. V. Parekh ◽

R. Mernaugh

Keyword(s):

Lung Cancer ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

Cancer Cell Lines ◽

Normal Lung ◽

Antibody Library ◽

Lung Cancer Cell ◽

Cell Lysates

e22137 Background: Lung cancer is the most common cause of cancer-related deaths in the world. There is a critical need for new strategies of early lung cancer detection. The identification of tumor-associated antigens and corresponding antibodies is one approach to discovery of diagnostic biomarkers. We used a large phage-displayed recombinant antibody library and normal human lung epithelial and non-small cell lung cancer cell lines to select for and identify recombinant antibodies specific for proteins expressed, or over-expressed, in lung cancer. Methods: The antibody library was used to select for recombinant scFv antibodies reactive with proteins present, or aberrantly expressed, in non-small cell lung cancer cell lines (A549, H549, H157, H23) in comparison to normal lung cell lines (BEAS-2B, 16-HBE, KT). Soluble scFv antibodies were obtained through 2 rounds of phage antibody cross-absorption (on normal cell lines) and selection (on non-small lung cancer cell lines). Soluble scFv were assayed by a high-throughput fluorometric microvolume assay technology (FMAT) against normal and cancer lung cell line proteins. ScFv antibodies selected by FMAT were evaluated further with Western blot-based assays. Results: More than 100 scFv antibodies identified by FMAT bound preferentially to proteins in lung cancer. Of these, 46 scFv were assayed by a high throughput Western slot blot immunoassay against pooled normal lung and lung cancer cell lysates. Eight scFv were assayed in Western blot against individual lung normal and non-small lung cancer cell line lysates. Four of these demonstrated differential binding to normal and cancer cell lysates. Conclusions: In summary, we were able to detect cancer-associated antigens in lung cancer cell lines using a phage display antibody library. In combination with high-throughput fluorescent and Western blot assays, four unique scFv antibodies were selected that differentially bound to normal and lung cancer cell lysates. These scFv will be tested as candidate biomarkers of lung cancer in independent tissue and serum samples from patient with and without lung cancer to determine utility for use in lung cancer diagnosis. No significant financial relationships to disclose.

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Development of High-Throughput Quantitative Assays for Glucose Uptake in Cancer Cell Lines

Molecular Imaging and Biology ◽

10.1007/s11307-010-0399-5 ◽

2010 ◽

Vol 13 (5) ◽

pp. 840-852 ◽

Cited By ~ 19

Author(s):

Mohamed Hassanein ◽

Brandy Weidow ◽

Elizabeth Koehler ◽

Naimish Bakane ◽

Shawn Garbett ◽

...

Keyword(s):

Glucose Uptake ◽

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

Cancer Cell Lines

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Detection of Intracellular Granularity Induction in Prostate Cancer Cell Lines by Small Molecules Using the HyperCyt® High-Throughput Flow Cytometry System

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109335671 ◽

2009 ◽

Vol 14 (6) ◽

pp. 596-609 ◽

Cited By ~ 21

Author(s):

Mark K. Haynes ◽

J. Jacob Strouse ◽

Anna Waller ◽

Andrei Leitao ◽

Ramona F. Curpan ◽

...

Keyword(s):

Prostate Cancer ◽

Flow Cytometry ◽

Small Molecules ◽

Cell Lines ◽

Cancer Cell ◽

High Throughput ◽

Prostate Cancer Cell ◽

Cancer Cell Lines ◽

Phenotypic Change ◽

Prostate Cancer Cell Lines

Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt® high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of ~25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells. ( Journal of Biomolecular Screening. 2009:596-609)

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