Many anti-cancer drugs work through inducing DNA damage, thereby activating the tumour suppressor protein TP53, unleashing many cellular processes, including apoptotic cell death, cell cycle arrest, cellular senescence and coordination of DNA repair. Typically, only one particular outcome will predominate in a given cell. The factors which determine the cellular response that will prevail in a cell, and ultimately decide the fate of that cell are still unclear. Understanding the factors that determine the cellular response to wild-type TP53 activation is important to comprehend how particular cells (malignant and non-transformed) will respond to therapy and also what cellular responses are activated by TP53 in cells during neoplastic transformation.
To investigate the coordination of these responses, a panel of human cancer-derived cell lines and non-transformed murine cells were treated with the MDM2 inhibitor, nutlin-3a, that activates TP53 in a non-genotoxic manner. We assessed levels of the cellular processes using flow cytometric assays, and found that after TP53 activation, both the cell lines and non-transformed murine cells follow one predominant TP53-induced response.
A subset of cell types were further analysed by RNA sequencing to investigate TP53-induced gene expression changes. Analysis of thymocytes (predominantly undergo apoptotic cell death), and dermal fibroblasts (undergo cell cycle arrest and cellular senescence), revealed a set of differentially expressed genes that could contribute to the differences seen in their fates. The functions of the “hits” are currently being investigated.
Further work will expand the RNA sequencing data set to include more diverse cell types and use oncogene expression to activate TP53 in the manner it is stimulated by oncogenic stress in cells undergoing neoplastic transformation. To use in parallel, we have also generated a panel of reporter mice in which one allele of genes critical for TP53-induced activation of apoptosis, cell cycle arrest, coordination of metabolism or DNA repair is replaced by a fluorescent reporter (td-Tomato for Puma, GFP for p21, Critine for Nprl3, BFP for Mlh1).