Abstract 5099: Automated rapid and accurate cell cycle analysis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA It is essential to have detailed information about compounds identified in screening campaigns during drug discovery and to know their influence on cell cycle progression or DNA repair. The discovery of Chromobodies® and advancements in High Content Imaging systems with improved optical properties and live cell capabilities has opened the door for new reliable and robust screening assays that provide these relevant high quality data. Chromobodies are a novel class of fluorescent antibodies, based on special heavy chain antibodies from camelids (VHHs). These unique nanoprobes can be transfected into cells as plasmid DNA similar to any conventional fusion protein. The cell expresses the fluorescent antibody-fragment thus enabling the non-invasive imaging of endogenous proteins and processes in real time. Therefore Chromobodies avoid any deleterious side effects from the traditional over-expression of a tagged protein. This is especially true for the essential components of the DNA replication, DNA repair and DNA methylation machinery: Proliferating Cell Nuclear Antigen (PCNA) and DNA Methyltransferase 1 (Dnmt1) . It has been shown that high over-expression of theses proteins can have adverse effects on normal progression through S phase. In this study we present a new method to evaluate cell cycle and DNA methylation. We developed Chromobodies directed against PCNA and Dnmt1. Transfecting these Chromobodies into cells circumvents the laborious and error-prone staining procedure with conventional antibodies. We demonstrate the flexible use of the Cell Cycle Chromobody cell line in conjunction with different automated high content imaging platforms. We show dose-dependent cell cycle inhibition caused by a number of anti-cancer drugs, DNA damage and apoptosis inducing compounds. Multiple analysis protocols enable the fast and accurate analysis with quantitative information on each sub phase of DNA replication for each individual cell. Moreover, by continuing to film cells, the exact length the cell cycle stages can also be extrapolated, thereby providing detailed temporal information on G1, S, G2 and M phase. Citation Format: Larisa Yurlova, Jacqueline Gregor, Oksana Sirenko, Evan Cromwell, Kourosh Zolghadr. Automated rapid and accurate cell cycle analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5099. doi:10.1158/1538-7445.AM2014-5099