Mapping P53 Alterations in Metabolism upon DNA Damage using the Phasor/FLIM and Number & Molecular Brightness Analysis

Abstract

p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migation and repair. If cells become stressed due to DNA damage, p53 will induce genes that trigger cell cycle arrest and/or apoptosis. p53 has a dual role in promoting oxidative phosphorylation (oxsphos) and glycolysis upon cellular stress. However, the metabolic function of p53 has not been fully explored. p53 metabolic activities may play an important role in normal growth, development, and tumor suppression, it might also be misused to help promote, rather than hinder, tumor development. To further assess p53 activity, we investigated the effect of metabolic changes under the same conditions and tested if the concentration of p53 influences the balance between apoptosis and DNA repair. We used the fluorescent lifetime imaging microscopy (FLIM) phasor approach to detect changes in oxsphos and glycolysis. Overall our data show the formation of p53 protein near the nucleolus and in areas near the inner regions of the nucleus. We also show that upon DNA damage the concentration of p53 increases by a factor of two independent of initial expression levels. The FLIM/Phasor data show that low concentrations of p53 is enough to trigger glycolytic response in the nucleus of the cells upon DNA damage with Cisplatin and under high expression levels, a new lifetime phasor cluster is detected. This new lifetime correlates with dead phenotype cells and may be a new “apoptotic” lifetime signal. In addition, we are able to visualize the sub-nuclear spatial localization of p53 tetramers upon DNA damage with cisplatin and obtain spatial-temporal malls of cellular localization and dynamics. This work is supported in part by NIH grants P50 GM076516 and P41 GM103540.