Abstract A21: RISCI: A high-throughput screening platform for investigating apoptosis and toxicity signaling.

Abstract

Abstract Apoptosis signaling is fundamental to disease progression and therapeutic success. Our group have developed a high-throughput genetic screening platform, named RISCI, with a daily throughput of 1,536 unique clones. By combining the wealth of genomics data with RISCI results, we attempt to identify new regulators of apoptosis signaling networks and hope that this approach offers insights into the regulation of compound efficacy and disease progression. Nanotechnology has been garnering immense interest, with potential applications for drug delivery and cancer treatments. However, the implementation of this new technology is hampered by the current limited knowledge. Some of these nanoparticles, in particular those at the lower size range, are cytotoxic due to increased surface area to volume ratio that enhances catalysis activity. The RISCI screening platform comprises a pair of custom-made robots handling DNA isolation and transfection, respectively. Ultrapure plasmid DNA is isolated and co-transfected with a beta-galactosidase reporter plasmid using an in-house developed protocol based on polyethylenimine polymer. The genes in our human cDNA library (NITE), a collection of approximately 30,000 full-length and sequenced genes, are systematically screened for causing membrane permeability. The integrity of the plasma membrane in apoptotic cells is damaged, allowing for the entry and turnover of the substrate to generate a colorimetric signal. Lysis with Triton after an initial measurement facilitates the assessment of transfection efficiency and readout normalization. Apoptosis-inducing candidates are then identified from a positive signal ratio. The use of silica nanoparticles extends the capacity of the RISCI screen to discover modulators of its toxicity. Initial results suggest that one of our apoptosis inhibitors is able to inhibit the cell death caused by these nanoparticles. Such an effect can also be assayed using the screen assay system, allowing for the identification of additional apoptosis inhibitors and sensitizers. While inhibitors are selected using a low signal ratio upon treatment with cytotoxic agents such as silica nanoparticles, genes sensitizing the cells to apoptosis upon treatment are highlighted by an increased signal ratio. A previous screen using a mouse cDNA library assayed in excess of 100,000 redundant clones and yielded 74 verified apoptosis inducers, including Orctl3, which was shown to have tumor-specific activity. The NITE library has significantly less redundancy, and current progress of approximately 4,000 clones identified 48 inducer candidates. The current discovery rate of 1.2% is much higher than previous attempts due to the reduced redundancy. These candidates will be subjected to further apoptosis assays such as Roche Nucleosome ELISA kit and PARP cleavage. Upon completion of the screen, 350–500 apoptosis inducing candidates are expected to be identified. Data are also being analyzed for inhibitors and sensitizers identification. The list of apoptosis inducers and inhibitors, when combined with the available microarrays and “omics” data, will allow us to identify new drug targets if the specific gene is implicated in a degenerative disease or cancer. The data from the nanoparticles screen can also identify new biomarkers that are specific to nanoparticle toxicity as part of a risk assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A21.