Selective modulation of the cyclin B/CDK1 and cyclin D/CDK4 complexes during in vitro human megakaryocyte development

Mammalian megakaryocyte development is characterized by a progressive accum ulation of cells exhibiting a polylobated nucleus with a polyploid DNA cont ent. In this study human megakaryocytes were obtained from CD34(+) haemopoi etic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO), Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the br eakdown of the nuclear envelope, and the progressive chromatin condensation , all aspects characteristic of mitosis. At both indirect immunofluorescenc e and Western blot analyses, cyclin B and its related cyclin-dependent kina se (CDK)1, which forms the mitosis promoting factor (MPF). showed an increa sed expression in maturating megakaryoblasts and megakaryocytes (day 8 of c ulture) with respect to freshly isolated CD34(+) progenitors. This expressi on tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete , mitotic process, and that cyclin D/CDK4 probably plays a crucial role thr oughout megakaryocytopoiesis.