Abstract
Abstract STAT3 which is a member of the family of signal transducers and activators of transcription is an oncogene and it is constitutively activated in numerous cancer cell lines and human cancers including breast, lung, head and neck, liver, and pancreas. Another STAT family member STAT1 possesses cancer-inhibitory properties and once activated may promote apoptosis in tumor cells. Thus STAT3 has been identified as a promising drug discovery target for many cancers. Ideally the potential inhibitors of STAT3 should be very selective and should not inactivate STAT1. To better address these cancer-research important genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via Zinc-Finger Nuclease (ZFN) mediated homologous recombination in cancer derived cell lines. ZFN technology is a fast and reliable way to manipulate the genome in a targeted fashion. ZFNs are synthetic proteins engineered to bind DNA at a sequence-specific location and create a double strand break (www.compozrzfn.com). The cell's natural machinery repairs the break in one of two ways: non-homologous end joining or homologous recombination. Utilizing the homologous recombination pathway that ZFNs induce, we successfully inserted the FP transgenes into the desired target locations - at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. Three cell lines were created, two with the single STAT tagged and a third with both STATs tagged. Integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins in the A549 cell lines. Functional analyses of knock-in cell lines indicated that both STAT3 and STAT1 native gene regulation is conserved resulting in normal levels of protein expression and preservation of protein function. When stimulated with IL-6 or INF-γ, A549 cells showed fast and robust nuclear translocation of RFP-STAT3 or STAT1-GFP respectively. Cell pre-incubation with a known specific STAT3 inhibitors showed that STAT1-GFP, INF-γ-induced translocation was not impaired. Thus, these cell lines provide a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors while ensuring that the STAT1 pathway is not affected. This approach of zfn-tagging endogenous genes could be applied to other cancer targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-506. doi:1538-7445.AM2012-LB-506
Published Version
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