Casein Kinase II Inhibition in MEG-01 Cell Line Results in Apoptosis, Megakaryocytopoiesis and Functional Platelets Release.

Abstract Megakaryocytes are polyploid cells, originating from hematopoietic stem cells in the bone marrow. Megakaryocytopoiesis is the process of production of anucleated cells, the platelets, from megakaryocytes. Megakaryocytes undergo endomitosis and maturation to the stage of proplatelets bearing megakaryocytes, which fragment to give rise to platelets. Platelets are vital for both maintaining normal hemostasis and for the response of the human body to trauma. Apoptosis is a genetically programmed and evolutionary conserved mechanism through which the normal development and tissue homeostasis are maintained. The platelets production is the result of constitutive apoptosis of megakaryocytes. Casein kinase II (CKII) is a pleiotropic, ubiquitous ectokinase that phosphorylates Serine, Threonine and Tyrosine amino acid residues. CKII has two catalytic subunits, α and α’ and two regulatory β subunits. The upregulation and hyperactivity of CKII has a general anti-apoptotic, pro-survival function in different overproliferative and carcinogenic processes. In chronic myelogenous leukemia (CML), specifically, CKII upregulation and hyperactivity induces stem cells increased proliferation. This results in bone marrow and circulating high blast levels and abnormal blood cells counts (trombocytosis, leukocytosis). We used the megakaryoblastic CML cell line, MEG-01, to study the effect of the inhibition of CKII, by CKII α subunit specific inhibitors, on platelet formation and cell proliferation. MEG-01 cells, which are defined as cytokine independent, are unresponsive to thrombopoietin and interferon. Incubation of MEG-01 cells with two different CKII inhibitors results in proliferation arrest, megakaryopoiesis, apoptosis and megakaryocytopoiesis. Platelet release from MEG-01 cells strongly depends on inhibitors concentration and treatment length. The platelets obtained from MEG-01 cells, following incubation with CKII inhibitors, get activated when stimulated by agonists, like phorbol-12-mystrate-13-acetate (PMA) and the peptide TRAP. These agonist-activated platelets undergo shape change and expose P-Selectin as a consequence of their activation. The platelet population stains positive for GpIIb/IIIa complex. Small size and negative staining for propidium iodide clearly distinguish platelets from megakaryocytes. In conclusion, we found a novel activator of platelet release from megakaryocytes. PMA, used as a megakaryocytopoiesis inducer, is known to stimulate release of functional platelets from MEG-01 cells, with normal blood platelet morphology. However, PMA, a very potent tumor promoter, induces cutaneous squamous cell carcinoma in mice. Our findings are important and demonstrate that platelets release from MEG-01 cells is a form of apoptosis that can be initiated following specific inhibition of CKII α subunit. Therefore, the processes of megakaryocytopoiesis and megakaryopoiesis are controllable through the fine-tuning of apoptosis. These results are original and of high physiological relevance since the high blast cell percentage can be decreased by proliferation arrest and differentiation. So the malignant processes in this type of leukemia can be stopped by controlling the stem cell number and their proliferation rate following inhibition of CKII α subunit. Thus, regulation of CKII α catalytic subunit may play a very important role in the future treatment of patients with CML that are resistant to the currently available treatments.