Role of Casein Kinase 2 in Platelets Release from Megakaryocytes.

Abstract Megakaryoblasts are precursors of platelets. Megakaryoblasts first differentiate to the stage of megakaryocytes. Megakaryocytes mature by increasing their size and by undergoing nuclear endoreplication and cytoplasmic maturation. Mature megakaryocytes form pseudopodia and reach pro-platelet bearing stage. Proplatelets bearing megakaryocytes fragment to give rise to platelets, through the process of thrombocytopoiesis. Both the thrombocytopoiesis and the megakaryocytopoiesis processes are linked to the constitutive apoptosis of megakaryocytic cells. The BCR/ABL oncoprotein is the result of a chromosomal translocation known as Philadelphia positive chromosome (Ph+). This is the main cause of several myeloproliferative disorders, like chronic myelogenous leukemia (CML). In CML the rate of platelet production is abnormal due to malignant megakaryoblast overproliferation. Casein kinase 2 (CK2) plays a crucial role in CML. CK2 was found to interact with BCR/ABL and to modulate its function. CK2 is also involved in many pathways downstream of BCR/ABL. CK2 acts on apoptotic pathways and also in the blood coagulation cascade. We studied the effect of casein kinase 2 alpha subunit (CK2α) inhibition in a megakaryoblastic cell line from a CML patient in blast crisis (MEG-01). We found that these inhibitors induce proliferation arrest while maintaining a steady cell number for a period of one week. Treated cells grew at a lower and constant rate than the non-treated ones, which are extremely prolific. Apoptosis of MEG-01 was induced by CK2 inhibitors, and this phenomenon was dose and time dependent. No necrosis was detected in the presence of the inhibitors, demonstrating that such compounds are not cytotoxic. Treatment of MEG-01 cells with CK2 inhibitors resulted in cells with apoptotic features like blebbing and specific apoptotic DNA fragmentation. Interestingly, these treatments lead to stimulation of megakaryocytopoiesis and thrombocytopoiesis processes in MEG-01 megakaryoblasts. In the presence of CK2 inhibitors megakaryocytes matured to the pro-platelets bearing stage. Platelets were released through rupture, following cytoplasmic fragmentation and nuclear extrusion. Thrombocytopoiesis due to the use of CK2 inhibitors occurred both in suspension as well as with MEG-01 cells grown on a fibronectin matrix. Platelets obtained following these treatments were harvested and analyzed for function. Such platelets were identified as anucleated cells by flow cytometry and microscopy techniques using DAPI and PI staining. These platelets were found to undergo shape change in response to various agonists (human thrombin, TRAP, ADP, PMA, fibronectin). MEG-01-derived platelets were also found to stain positive with CD41a antibody (αIIbβ3) as determined by flow cytometry using a monoclonal antibody specific to the receptor. Following agonist activation, the platelets expose phosphatidylserine and P-Selectin. The specific use of PAC-1, an antibody that only recognizes the activated form of αIIbβ3, together with fibrinogen binding studies, demonstrated that the MEG-01-derived platelets can be activated. Fibronectin, RGDS and EDTA inhibited activation of MEG-01-derived platelets. Addition of human thrombin (5 U/ml) and fibrinogen to these platelets resulted in a stable clot. These findings suggest that CK2 is involved in MEG-01 differentiation, platelets production, and activation. Thus, by using a CK2 inhibitor we have successfully stopped the abnormal proliferation of a transformed cell line and reversed the path towards its normal function.