Regulation of ES cell differentiation by functional and conformational modulation of p53.

Embryonic stem (ES) cell lines were used to examine the role of p53 during in vitro differentiation. Undifferentiated ES cells express high levels of p53 exclusively in the wild-type conformation, immunoprecipitable by monoclonal antibody PAb246, and p53 was found to be functionally active as determined by its ability to bind DNA specifically and to activate transcription of target genes. Differentiation in vitro resulted in a decrease in the levels of p53 and in a shift in its conformational status to the mutant form, detectable by monoclonal antibody PAb240, with a concomitant loss of functional activity. The presence of functional p53 in the undifferentiated ES cells renders them hypersensitive to UV irradiation, whereas the differentiated cells were resistant to UV treatment. ES cells lacking p53 exhibit enhanced proliferation in both the undifferentiated and differentiated state, and apoptosis accompanying differentiation was found to be reduced. Furthermore, wild-type ES cells undergoing apoptosis expressed functional p53. Expression of the temperature-sensitive p53val135 mutant in wild-type ES cells resulted in a reduction of apoptosis accompanying differentiation when it adopted a mutant conformation at 39 degrees C. These data demonstrate that functional inactivation of p53 allows differentiating cells to escape from apoptosis, and suggest that the conformational switch could regulate the inactivation process.