Early down-regulation of Bcl-xL expression during megakaryocytic differentiation of thrombopoietin-induced CD34+ bone marrow cells in essential thrombocythemia

Abstract

BACKGROUND AND OBJECTIVES: Essential thrombocythemia (ET) is a chronic myeloproliferative disorder with abnormal megakaryocyte/platelet production. Recent studies have found that Bcl-xL, as a member of the bcl-2 family of proteins that inhibit apoptosis, is essential in megakaryocytic differentiation. In this study the expression of Bcl-xL was evaluated during megakaryocytic differentiation in ET patients. DESIGN AND METHODS: To study the role of Bcl-xL in megakaryocyte differentiation, we evaluated the effect of small interfering RNA (siRNA) on the expression of Bcl-xL. CD34+ cells from patients with ET, chronic myeloid leukemia (CML), polycythemia vera (PV) and normal individuals were cultured in serum-free medium supplemented with thrombopoietin (TPO). Immunocytochemical staining and flow cytometric analysis were used to evaluate the Bcl-xL expression during megakaryocytic differentiation of CD34+ cells. RESULTS: When exposured to si-Bcl-xL, the percentage of K562 cells induced into megakaryocytes in 72 hours was lower than the corresponding percentage of control cells. CD41a+ cells from the three groups of patients and the control group were cultured. At day 10, the percentage of Bcl-xL- cells in CD41a+ cells from ET patients was 61.0+/-28.1%, which was significantly higher than that from patients with CML (2.5+/-20.9%), PV (33.6+/-10.0%) or control subjects (15.1+/-13.0%).] INTERPRETATION AND CONCLUSIONS: These results demonstrate that Bcl-xL is down-regulated early during in vitro differentiation of megakaryocytes from ET patients; this might reflect an early entry of megakaryocytes into a degenerating mature stage.