N3 induced reprogramming and ShWT1 knock-down in DSRCT cell lines

The object of this study is to identify the molecular signature underlying the therapeutic potential of N3-induced growth arrest in Desmoplastic Small Round Cell Tumor (DSRCT). The molecular hallmark of DSRCT is the EWS-WT1 fusion protein formed by chromosomal translocations found in DSRCT. The N-terminal transactivation domain of EWS is fused to the C-terminal DNA binding domain of WT1, creating an oncogenic transcription factor. We have recently discovered that addition of N3 supplement to growing DSRCT cells leads to a rapid cessation of cell growth. To identify the effect of N3 induced growth arrest, U2OS (control) or DSRCT cells will be cultured in regular media or media containing N3 supplement. Three independent experiments will be performed for each cell line with indicated media. A second set of experiment will be to examine the effects of depleting the EWS/WT1 oncogene with a short hairpin ShWT1 or shControl in DSRCT cell lines grown in the absence or presence of N3 Supplement. Total RNA will be isolated and analyzed by microarray analysis. The untreated (no N3) will serve as a reference point for the first experiment, and the shControl transfected samples will serve as a reference for the second experiment. We will probe Affymetrix human genome U133 plus 2.0 arrays with total RNA samples. For all experiments, we will use total RNA isolated in triplicate (biological triplicates).