Expression profiling of p53 transcriptional target genes as a predictor of cell fate following non-genotoxic activation of p53 by MDM2 inhibitors (360G-Wellcome-211752_Z_18_Z)

The purpose of the proposed project is to investigate the differences in p53-dependent gene expression between cells that undergo growth inhibition and senescence in response to p53 activation compared with those that undergo apoptosis. These alternative cell fates, which are cell type and context dependent, have important roles in a range of disease states and responses to therapeutic intervention, but the underlying mechanisms are poorly understood. We have identified a panel of cell lines that epitomise these alternative cell fate responses to p53 activation, which we will investigate by using MDM2-p53 binding antagonists to activate p53 directly and specifically, without DNA damage. In addition, we will use agents which demethylate DNA to test whether pro-apoptotic genes can be re-activated in those cell lines in which these genes appear to be silenced. Techniques used to investigate the downstream consequences of p53 activation will include flow cytometry, enzymatic apoptosis and senescence assays, Western immunoblotting and multiplex qRT-PCR gene expression profiling.