We examined the effects of K-252a on polyploidization and differentiation of a human megakaryocytic cell line, Meg-J. Although K-252a is known to induce polyploidization in some non-haematological cell lines ( Usui et al, 1991 ), this is the first report concerning its effect on megakaryocytic cells. K-252a induced Meg-J cells to form polyploid cells, which were accompanied by the morphological differentiation and increases in the levels of GPIIb/IIIa and GPIb expression. It has been suggested that DNA synthesis is a prerequisite not only for polyploidization but also for the expression of megakaryocytic markers such as GPs in TPA-treated megakaryocytic differentiation ( Murata et al, 1991 , 1993; Yoshino et al, 1996 ). In our system, such DNA synthesis without cell division may also have induced increased levels of megakaryocytic markers.
K-252a also induced polyploidization and increased the levels of surface expression of GPIIb/IIIa and GPIb on CMK, MEG-01, UT-7, HEL and K562, all of which are known to have megakaryocytic features or the capacity to differentiate into cells of the megakaryocytic lineage (data not shown). However, K-252a did not induce obvious polyploidization of non-megakaryocytic cell lines such as MOLT-4 (a human lymphoid leukaemia cell line) and HL-60 (a human myeloid leukaemia cell line) (data not shown). These results suggest that, even in haemopoietic cells, polyploidization-inducing activity of K-252a is limited to cells with potential megakaryocytic features. This K-252a-induced polyploidization and differentiation of Meg-J cells was enhanced by simultaneous addition of TPO. This might have been due to the increasing survival of Meg-J cells by TPO.
Given the potential importance of G2/M regulation in polyploidization, we performed detailed analyses of expression and function of cyclin B1 and cdc2 which constitute critical kinase involved in G2/M transition in K-252a-treated Meg-J cells. It has been demonstrated that dephosphorylation of Thr-14 and Tyr-15 of cdc2 during the activation at the prophase/metaphase transition occurs in two steps; first, Thr-14 is dephosphorylated, and then Tyr-15 is dephosphorylated. The Thr-14 dephosphorylated and Tyr-15 phosphorylated form of cdc2 still retains significant kinase activity ( Borgne & Meijer, 1996). In K-252a-treated Meg-J cells, total levels of cdc2 were transiently increased at 12 h after release from the hydroxyurea block. The intermediate migrating form (Thr-14 dephosphorylated and Tyr-15 phosphorylated form) and the fastest migrating form (Thr-14 and Tyr-15 dephosphorylated form), both of which were active as cdc2 kinase, were clearly detected. Furthermore, cdc2 kinase activity was also transiently increased at 12 h after release from hydroxyurea block. These findings indicated that K-252a did not block the dephosphorylation of Thr-14 and Tyr-15 (and also phosphorylation of Thr-161), and did not interfere with the transient increase of cdc2 kinase activity at 12 h after releasing the hydroxyurea block. On the other hand, K-252a reduced the expression of total cdc2 thereafter, especially the phosphorylated Tyr-15 form, indicating that cdc2 was not newly synthesized. In addition, expression of cyclin B1 started to reduce after 12 h. These changes in cdc2 and cyclin B1 expression resulted in decreased cdc2 kinase activity. Polyploidization began to occur more than 48 h after release from the hydroxyurea block in K-252a-treated cells ( Figs 3C and 5A), when the levels of cdc2 and cyclin B1 became markedly decreased. Hence, the elevations and subsequent decreases in cdc2 and cyclin B1 levels (as well as cdc2 kinase activity) preceded K-252a-induced polyploidization. In some megakaryocytic cell lines such as MEG 01, HEL and U937, treatment of cells with TPA induced inhibition of cdc2 kinase activity, followed by polyploidization. In this case, levels of cdc2 and cyclin B1 did not change, and lack of cdc2 kinase activity was due to the down-regulation of cdc25C phosphatase ( Garcia & Cales, 1996). It has also been reported that lack of cdc13 (cyclin B) induces entry of Sch. pombe cells into an endoreplication cycle ( Hayles et al, 1994 ). Furthermore, overexpression of rum1, which appears to be a cdc2 inhibitor, causes Sch. pombe cells to enter an endoreplication cycle ( Moreno & Nurse, 1994). These findings indicate that inhibition of cdc2 kinase activity appears to be a key event for endoreplication (or polyploidization). Intriguingly, our results obtained in megakaryocytic cells indicates that transient elevation of cdc2 kinase occurred before progressive decrease of the kinase activity. Recently, it was reported that normal polyploid megakaryocytes enter mitosis and step into anaphase. In such cells, accumulation of both cyclin B1 and cdc2 was clearly detected during early mitosis ( Nagata et al, 1997 ). Based on the generally accepted idea that activation of cdc2 kinase is an essential prerequisite to initiate mitosis, these findings indicate that, in normal megakaryocytes, elevation of cdc2 kinase activity occurs in the process of polyploidization, as has been demonstrated in K-252a-treated Meg-J cells. Accordingly, our system shown here may be an useful model to further elucidate mechanisms underlying normal megakaryocytic polyploidization.