Increased D-type cyclin expression together with decreased cdc2 activity confers megakaryocytic differentiation of a human thrombopoietin-dependent hematopoietic cell line

At the late phase of megakaryocytopoiesis, megakaryocytes undergo endomitos is, which is characterized by DNA replication without cell division. Althou gh a number of cell. cycle regulatory molecules have been identified, the p recise roles of these molecules in megakaryocytic endomitosis are largely u nknown. In a human interleukin-3-dependent cell line transfected with the t hrombopoietin (TPO) receptor c-mpl (F-36P-mpl), either treatment with TPO o r the overexpression of activated ras (Ha-Ras(G12V)) induced megakaryocytic maturation with polyploid formation, We found that TPO stimulation or Ha-R as(G12V) expression led to up-regulation of cyclin D1, cyclin D2, and cycli n D3 expression. In addition, expression levels of cyclin A and cyclin B we re reduced during the total course of both TPO- and Ha-Ras(G12V)-induced me gakaryocytic differentiation, thereby leading to decreased cdc2 kinase acti vity. Neither the induced expression of cyclin DI, cyclin D2, or cyclin D3 nor the expression of a dominant negative form of cdc2 alone could induce m egakaryocytic differentiation of F-36P-mpl cells. In contrast, overexpressi on of dominant negative cdc2 together with cyclin D1, cyclin D2, or cyclin D3 facilitated megakaryocytic differentiation in the absence of TPO. These results suggest that both D-type cyclin expression and decreased cdc2 kinas e activity may participate in megakaryocytic differentiation.