Tumor Suppressor Protein p53 Affects Megakaryocytic Maturation: In Vivo and Ex Vivo Post-Translational Modification Studies

BACKGROUND AND HYPOTHESIS: Tumor suppressor p53 responds to cellular stress and elicits arrest of DNA synthesis and/or apoptosis. We have recently demonstrated that p53 is activated during Megakaryocytic (Mk) differentiation in both human primary cultured Mk cells and in the CHRF megakaryoblastic cell line. We also showed that miRNA-mediated down-regulation of p53 in CHRF cells results in higher Mk ploidy by means of enhancement of DNA synthesis and deferment of apoptosis. Additionally, we have shown that down-regulation of p53 affects its transcriptional activity and modulates the level of expression of its canonical transcriptional targets (

METHODS: Multiparametric flow cytometry (FC) is a powerful technique to investigate cell signaling pathways permitting the simultaneous interrogation of single cells and detection of several post-translationally modified p53 proteins. We used FC to examine the in vivo levels of p53 in mouse bone marrow (BM) Mk cells and compare such levels to p53 levels in non-Mk BM cells and granulocytes (G cells) in particular (Gr-1+ cells). In addition we employed FC to examine the levels of total p53, p53 phosphorylated on serines 15, 37 and 46 and p53 acetylated on lysine 382 in Mk (CD41+) cells generated from culture of human mobilized peripheral blood-derived CD34+ cells with thrombopoietin (Tpo). Finally, we compare the levels of p53 in ex vivo generated Mk cells with those found in isogenic culture-derived G cells.

RESULTS: In mice, we found that p53 is expressed at higher levels in CD41+ cells in the BM than other BM cells and Gr-1+ cells in particular. We established also that high p53 levels in Mk cells is not due to the large size of polyploid Mk cells, since it holds for 2N cells of either lineage. During ex vivo generated megakaryopoiesis, we observed a large increase in the percentage of polyploid Mk cells between days 7 and 9 of Tpo culture and by day 11 most Mk cells have become apoptotic. Total p53 levels increased continuously in Mk cells up until the end of Tpo culture (day 11). p53 levels were always substantially higher in Mk cells than in isogenic CD15+ G cells. Using immunofluorescence microscopy, we demonstrated that while p53 is low and mostly cytoplasmic in CD34+ cells, it translocates to the nucleus upon Mk differentiation. Consensus-DNA binding of p53 (by EMSA assays using nuclear extracts) continued to increase in CD41+ cells between days 5 and 9 of culture and eventually dropped at the end (day 11). The level of phosphorylated or acetylated p53 was higher in CD41+ than in CD34+ cells or in the CD41− cells of the culture. The sole exception was p53 phosphorylated on serine 46, which was expressed lower in CD41+ than in CD41− cells, but still higher than CD34+ cells. Phosphorylated p53 peaked around day 7 of Tpo culture in both absolute levels and as a fraction of total p53. When nicotinamide, a Sir2 deacetylase inhibitor, was added to increase ploidy and cell size (

CONCLUSIONS: We conclude that p53 is activated during the course of Mk differentiation both in vivo and ex vivo and that the levels of p53 are predominantly high in Mk cells when compared to other isogenic hematopoietic cells. Taken together, these data support a model that activation of p53 occurs early in Mk differentiation through acquisition of specifically regulated posttranslational modifications and that the transcriptional activity of p53 is attenuated at a late stage of differentiation serving to increase DNA synthesis and polyploidization, which is in line with our hypothesis about the role of p53 in megakaryopoiesis.