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 du ring in vitro differentiation. Undifferentiated ES cells express high levels of p53 exclusively in the wild-type conformation, immunoprecipi table by monoclonal antibody PAb246, and p53 was found to be functiona lly active as determined by its ability to bind DNA specifically and t o activate transcription of target genes, Differentiation in vitro res ulted in a decrease in the levels of p53 and in a shift in its conform ational status to the mutant form, detectable by monoclonal antibody P Ab240, with a concomitant loss of functional activity, The presence of functional p53 in the undifferentiated ES cells renders them hypersen sitive to UV irradiation, whereas the differentiated cells were resist ant to UV treatment, ES cells lacking p53 exhibit enhanced proliferati on in both the undifferentiated and differentiated state, and apoptosi s accompanying differentiation was found to be reduced, Furthermore, w ild-type ES cells undergoing apoptosis expressed functional p53. Expre ssion of the temperature-sensitive p53val135 mutant in wild-type ES ce lls resulted in a reduction of apoptosis accompanying differentiation when it adopted a mutant conformation at 39 degrees C, These data demo nstrate that functional inactivation of p53 allows differentiating cel ls to escape from apoptosis, and suggest that the conformational switc h could regulate the inactivation process.