Cell confluency affects p53 dynamics in response to DNA damage

Abstract

The tumor suppressor protein p53 plays a key role in the cellular response to DNA damage. In response to DNA double-strand breaks (DSB), cultured cells exhibit oscillations of p53 levels, which impact gene expression and cell fate. The dynamics of p53 in vivo have only been studied in fixed tissues or using reporters for p53's transcriptional activity. Here we established breast tumors expressing a fluorescent reporter for p53 levels and employed intravital imaging to quantify its dynamics in response to DSB in vivo. Our findings revealed large heterogeneity among individual cells, with most cells exhibiting a single prolonged pulse. We then tested how p53 dynamics might change under high cell confluency, one factor that differs between cell culture and tissues. We revealed that highly confluent cultured breast cancer cells also show one broad p53 pulse instead of oscillations. Through mathematical modeling, sensitivity analysis, and live-cell imaging, we identified low levels of the phosphatase Wip1, a transcriptional target and negative regulator of p53, as a key contributor to these dynamics. Because high cell confluency better reflects the microenvironment of tissues, the impact of cell confluency on p53 dynamics may have important consequences for cancerous tissues responding to DNA damage-inducing therapies.