Role of p2l(CiP1/Waf1) in cell-cycle exit of endomitotic megakaryocytes

The cyclin-dependent kinase inhibitor p21(Waf-1/Cip-1) is expressed at high level during megakaryocyte differentiation, but its precise function remai ns unknown. In this study, it is confirmed that p21 was expressed at a high level in hypoploid (2N and 4N) and polyploid (at least 8N) human megakaryo cytes derived from CD34(+) cells. A high expression of p27(Kip1), p16, cycl in E, and cyclin D3 was also found in both populations associated with a hy pophosphorylated form of retinoblastoma protein, suggesting that the majori ty of hypoploid and polyploid megakaryocytes are Gl-arrested cells. As huma n megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to the murine model. The modal ploidy of megakaryocytes derived from lineage-negative cells was 32N, and an elevated expression of p21 was found in high-ploidy megakaryocytes. In addition, p21 and p27 were coexpressed in the majority of mature polyploid megakaryocytes. The p21 wa s detected by immunofluorescence in megakaryocytes derived from p53(-/-) mi ce, demonstrating a p53-independent regulation during megakaryocyte differe ntiation. Megakaryocytopoiesis of p21(-/-) mice was subsequently studied. N o marked abnormality in the ploidy of primary or cultured megakaryocytes wa s detected. Overexpression of p21 in p21(-/-) or normal murine megakaryocyt es and in human megakaryocytes showed in all these cases a marked inhibitio n in megakaryocyte polyploidization. In conclusion, while a reciprocal rela tion is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy pro file in normal megakaryocytes in vivo. However, high levels of its expressi on in cultured megakaryocytes arrest the endomitotic cell cycle. (Blood. 20 01;98:3274-3282) (C) 2001 by The American Society of Hematology.