Screen for modulators of DNA replication

Dorit Arlt
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

Ulrich Tschulena, Mamatha Sauermann, Nina Claudino, Angelika Duda
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. The whole process of data analysis is automated and performed in collaboration with the Bioinformatics and Modeling group. Reference: Sauermann 2007.

Assay to detect proteins impacting the cell cycle

Stephanie Bechtel, David Zhang, Ulrich Tschulena, Ioanna Keklikoglou, Anja Irsigler
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. Data analysis is performed in a collaboration with the Bioinformatics and Modeling group.

Screen for activators and repressors of P38 signaling

Stephanie Bechtel, Anja Irsigler, Angelika Duda, Ute Ernst, Ulrich Tschulena
Via stress activation, the P38 signal transduction pathway has impact on numerous biological processes, including inflammation, cell death, cell cycle control and cancer. We screened for genes influencing P38 MAP kinase signalling when knocked-down via RNAi using a human genome-wide siRNA library containing over 21,000 siRNA pools. A pathway-specific luciferase reporter cell line was transfected in 96-well format and assayed for reporter gene expression reflecting the activation status of P38 signalling. Data were processed in a high-throughput analysis pipeline comprising of multiple pre-processing, normalization and quality assessment steps, generating interactive HTML outputs. In particular, replicate measurements were summarized and any artificial plate and batch effects were removed. Thus, we acquired scored values of activation or inhibition in an unbiased and objective manner, and were able to audit critical steps of the analysis procedure. By these means, we could identify a number of genes/proteins with a modulating effect on the P38 signalling pathway. These include several kinases, membrane receptors, and transcription factors, some of which are known to be related with P38 signalling. Novel genes and proteins identified will be further validated and candidate genes functionally analysed in detail for their involvement in the cellular processes that are connected with P38 signal transduction.

Modulators of microRNA-21

Ulrich Tschulena, Nina Claudino, Esther Backes, Heike Wilhelm
MicroRNAs (miRNAs), endogenous small non-protein-coding RNAs, regulate many basic cellular processes including proliferation, differentiation and apoptosis. Recent evidence has shown that alterations in miRNA expression can also contribute to tumor growth by modulating critical genes. MiR-21 is upregulated in breast cancer, prostate cancer, glioblastoma and other cancers and has been proven to correlate with tumor stage and vascular invasion. To identify genes involved in the regulation of miR-21 we are screening a siRNA-library of 4,000 selected siRNAs, which were chosen based on their potential to regulate microRNA biogenesis, utilizing a robotic pipeline. For a primary screen, a target site for miR-21 has been cloned into the 3’-UTR of the luciferase reporter-gene. In this way, changes in miR-21 level after transfection of siRNAs result in changes of luciferase concentration. Hits in the primary screen are validated in a secondary screen, which detects changes in miR-21 concentration directly by using quantitative real time PCR. By these screens we aim to identify novel tumor-promoting or tumor-suppressing genes, which modulate tumor growth by regulating miRNAs.