Abstract A51: Development and initial characterization of isogenic cell lines stably overexpressing prostate cancer candidate genes

Microarray analyses of global changes in gene expression patterns have recovered a large number of genes, which are deregulated in prostate cancer, when compared to normal prostate tissue. In order to successfully translate these data into clinical intervention strategies, it is crucial to determine genes that truly contribute to tumor development (which may be referred to as ‘driver genes’) and to separate them from the majority of secondary anomalies in expression levels (‘passenger genes’). To obtain the missing functional information, we construct isogenic cell line libraries derived from commonly used prostate (RWPE‐1) and prostate cancer (LNCaP, PC‐3) cell lines and subject them to a range of functional assays. Using a system for rapid generation of stably‐transfected isogenic cell lines, that was developed in our group, we create a prostate cancer library in a two‐step procedure. At first, acceptor clones are generated from cancer and normal prostate cell lines by stable integration of a plasmid carrying a recombinase target sequence. Incorporation of a second expression plasmid with the gene of interest is then mediated by site‐specific recombination. This procedure allows a facilitated transgene integration into a predefined, transcriptionally active locus. It also minimizes the influence of genetic background, as clones generated within such a library differ among each other only by the presence of the gene of interest in the same chromosomal location. Therefore, in contrast to traditional knock‐in strategies, our system ensures that readily interpretable phenotypes are obtained and thereby provides a highly standardized resource for functional gene analysis. The technology is well suited for medium throughput and was designed to link data from high‐throughput genomic analyses with functional gene analysis in animal models. LNCaP and PC‐3 acceptor cell lines have been generated and characterized. Recombination of PC‐3 with several well‐described, cancer‐relevant genes under the control of an inducible promoter indicated the feasibility of the method. The next step will include initial screening of the library of roughly 100 recombinants for changes in the cell viability and proliferation. The most promising effectors from the primary screen will be followed with an expanded range of functional assays, addressing their impact on cell cycle, apoptosis and cell migration. This can then serve as a starting point for an individual characterization of the novel prostate cancer genes. Citation Information: Cancer Res 2009;69(23 Suppl):A51.