High-throughput cellular screens

Global microRNA level regulation of EGFR-driven cell-cycle protein network in breast cancer

The EGFR-driven cell-cycle pathway has been extensively studied due to its pivotal role in breast cancer proliferation and pathogenesis. Although several studies reported regulation of individual pathway components by microRNAs (miRNAs), little is known about how miRNAs coordinate the EGFRprotein networkona globalmiRNA(miRNome) level. Here,we combined a large-scalemiRNA screening approach with a high-throughput proteomic readout and network-based data analysis to identify which miRNAs are involved, and to uncover potential regulatory patterns. Our results indicated that the regulation of proteins by miRNAs is dominated by the nucleotide matching mechanism between seed sequences of the miRNAs and 30-UTR of target genes. Furthermore, the novel network-analysis methodology we developed implied the existence of consistent intrinsic regulatory patterns where miRNAs simultaneously co-regulate several proteins acting in the same functional module. Finally, our approach led us to identify and validate three miRNAs (miR-124, miR-147 and miR-193a-3p) as novel tumor suppressors that co-target EGFR-driven cell-cycle network proteins and inhibit cell-cycle progression and proliferation in breast cancer (Uhlmann 2012).

Time-resolved human RNAi screening for early regulators of cell proliferation

Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening. Reference: Zhang 2011.

Screen for modulators of DNA replication

Every chromosome is doubled in the DNA-synthesis, or S-phase, of the cell cycle before cells divide. Cell cycle checkpoints normally insure that DNA replication occurs only when conditions are favorable and cells are in a state where the process is working correctly. Deregulation of genes encoding cell cycle proteins can lead to uncontrolled proliferation which is a major cause for tumor formation. We have established a proliferation assay that is based on the incorporation of BrdU during S-phase, and identifies proteins that affect DNA replication when being overexpressed. The effect of YFP-fused proteins is detected through immunofluorescent staining of incorporated BrdU and flow cytometric analysis. Reference: Arlt 2005.

Cellular Assays and screens to detect proteins impacting apoptosis

Apoptosis is a cell suicidal mechanism that is characterized by a unique and highly controlled series of biochemical and morphological events. The activation of caspase-3 is a central and specific event in the process of apoptosis. This protein is a major effector or executioner protease, which cleaves several intracellular target proteins and ultimately leads to cell death once activated. We have developed a high throughput assay, which uses the activated endogenous caspase-3 as a marker for apoptosis. The activation of caspase-3 in response to ectopic overexpression or downregulation of endogenous proteins is detected with a specific antibody. Moreover, changes in caspase-3 concentration after activation of apoptosis with chemical inducers can be monitored in cells with inhibited or overexpressed proteins. Caspase-3-staining is carried out in a fully automated protocol, and the fluorescence intensities are measured using a flow cytometer with an integrated 96-well plate reader. Reference: Sauermann 2007.

Assay to detect proteins impacting the cell cycle

Cell division is a key process for all organisms. For a cell to divide it has to undergo different phases in the cell cycle, starting from growth in G1-phase via S-phase, in which the DNA is replicated and therefore DNA content is doubled, and then through G2-Phase leading to M-phase in which the cell divides. We have developed a rapid and robust DNA content assay to identify genes required for cell cycle progression. To this end, we use the fluorescent chemical compound 7-AAD which intercalates quantitatively in double-stranded DNA. The fluorescence intensities are measured using a flow cytometer with an integrated 96-well plate reader. 

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