Role of Tumor Suppressor p53 in Megakaryopoeisis, Platelet Formation and Platelet Function

Tumor suppressor p53 responds to cellular stress by eliciting cell cycle arrest and/or apoptosis. Despite the importance of cell cycle regulation and apoptosis in aspects of megakaryocytic maturation, such as endomitosis, leading to polyploidization, little is known about the role of p53 during megakaryopoeisis.;In megakaryocytic cultures of human CD34+ cells with thrombopoietin, p53 translocated to the nucleus and became activated by acquiring a set of post-translational modifications. However, in the late phase of megakaryocytic culture, which coincides with increased polyploidization, post-translationally modified p53 levels declined, indicating diminished activation of p53.;Knock-down of p53 in CHRF cells, a megakaryoblastic cell line, led to increased polyploidization by means of enhanced DNA synthesis and delayed apoptosis upon PMAinduced terminal megakaryocytic differentiation. p53 knock-down also led to a significant differential regulation of a number of genes related to cell cycle regulation, apoptosis and the cytoskeleton. Additionally, a set of genes coding for proteins that are essential in platelet functions was found differentially regulated upon p53 knock-down. The latter finding motivated us to examine the effect the absence of p53 has on platelet functional responses that determine hemostasis. Our studies have shown that isolated p53-/- platelets are less sensitive than p53+/+ platelets to agonist stimulation in binding fibrinogen and in secreting P selectin on their surface. This finding suggests a specific defect in inside-out signaling during p53-/- platelet activation.;p53-/- mice do not appear to have any defects in megakaryopoiesis and platelet production in vivo. However, ex-vivo cultured megakaryocytes from p53-/- mice achieve higher ploidy classes in part by exhibiting enhanced DNA synthesis during polyploidization. Administration of thrombopoeitin to p53-/- mice led to moderately increased ploidy of bone marrow megakaryocytes, but did not significantly affect platelet levels compared to p53+/+ mice. Moreover, antibody-induced thrombocytopenia did not reveal any differences in megakaryopoeisis and platelet production between p53+/+ and p53-/- mice. These findings indicate that loss of p53 affects ploidy during megakaryopoeisis mainly when the megakaryocytes are cultured ex-vivo and provide a new direction for efforts to increase Mk maturation, particularly the extent of polyploidization, in cultured megakaryocytes.