Role of p21(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 remains 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 megakaryocytes derived from CD34(+) cells. A high expression of p27(Kip1), p16, cyclin E, and cyclin D3 was also found in both populations associated with a hypophosphorylated form of retinoblastoma protein, suggesting that the majority of hypoploid and polyploid megakaryocytes are G(1)-arrested cells. As human 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 was detected by immunofluorescence in megakaryocytes derived from p53(-/-) mice, demonstrating a p53-independent regulation during megakaryocyte differentiation. Megakaryocytopoiesis of p21(-/-) mice was subsequently studied. No marked abnormality in the ploidy of primary or cultured megakaryocytes was detected. Overexpression of p21 in p21(-/-) or normal murine megakaryocytes and in human megakaryocytes showed in all these cases a marked inhibition in megakaryocyte polyploidization. In conclusion, while a reciprocal relation is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy profile in normal megakaryocytes in vivo. However, high levels of its expression in cultured megakaryocytes arrest the endomitotic cell cycle.
Baccini
V
V
INSERM U 362, Institut Gustave Roussy, Villejuif, France.
Roy
L
L
Vitrat
N
N
Chagraoui
H
H
Sabri
S
S
Le Couedic
J P
JP
Debili
N
N
Wendling
F
F
Vainchenker
W
W
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000032:analysis; D002454:Cell Differentiation; D002460:Cell Line; D056743:Cyclin D3; D019927:Cyclin E / Q000032:analysis; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology; D006801:Humans; D019394:Ki-67 Antigen / Q000032:analysis; D008533:Megakaryocytes / Q000737:chemistry / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008938:Mitosis; D011003:Ploidies; D011092:Polyethylene Glycols / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000032:analysis
D018952:Antigens, CD34; C534696:CCND3 protein, human; C493956:CDKN1A protein, human; C534697:Ccnd3 protein, mouse; C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D056743:Cyclin D3; D019927:Cyclin E; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D019394:Ki-67 Antigen; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C103117:polyethylene glycol-recombinant human megakaryocyte growth and development factor; D050760:Cyclin-Dependent Kinase Inhibitor p27; D011092:Polyethylene Glycols; D013926:Thrombopoietin