RESUMO
The development of episomally maintained DNA vectors to genetically modify dividing cells efficiently and stably, without the risk of integration-mediated genotoxicity, should prove to be a valuable tool in genetic research. In this study, we demonstrate the utility of Scaffold/Matrix Attachment Region (S/MAR) DNA vectors to model the restoration of a functional wild-type copy of the gene folliculin (FLCN) implicated in the renal cancer Birt-Hogg-Dubé (BHD). Inactivation of FLCN has been shown to be involved in the development of sporadic renal neoplasia in BHD. S/MAR-modified BHD tumor cells (named UOK257-FS) show restored stable FLCN expression and have normalized downstream TGFß signals. We demonstrate that UOK257-FS cells show a reduced growth rate in vitro and suppression of xenograft tumor development in vivo, compared with the original FLCN-null UOK257 cell line. In addition, we demonstrate that mTOR signaling in serum-starved FLCN-restored cells is differentially regulated compared with the FLCN-deficient cell. The novel UOK257-FS cell line will be useful for studying the signaling pathways affected in BHD pathogenesis. Significantly, this study demonstrates the suitability of S/MAR vectors to successfully model the functional expression of a therapeutic gene in a cancer cell line and will aid the identification of novel cancer markers for diagnosis and therapy.Molecular Therapy-Nucleic Acids (2013) 2, e115; doi:10.1038/mtna.2013.40; published online 13 August 2013.
RESUMO
The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into "immune deficient" mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome.
