Activation of p53 by nutlin leads to rapid differentiation of human embryonic stem cells.
p53 Is an important regulator of normal cell response to stress and frequently mutated in human tumours. Here, we studied the effects of activation of p53 and its target gene p21 in human embryonic stem cells. We show that activation of p53 with small-molecule activator nutlin leads to rapid differentiation of stem cells evidenced by changes in cell morphology and adhesion, expression of cell-specific markers for primitive endoderm and trophectoderm lineages and loss of pluripotency markers. p21 is quickly and dose-dependently activated by nutlin. It can also be activated independently from p53 by sodium butyrate, which leads to the differentiation events very similar to the ones induced by p53. During differentiation, the activating phosphorylation site of CDK2 Thr-160 becomes dephosphorylated and cyclins A and E become degraded. The target for CDK2 kinase in p53 molecule, Ser-315, also becomes dephosphorylated. We conclude that the main mechanism responsible for differentiation of human stem cells by p53 is abolition of S-phase entry and subsequent stop of cell cycle in G0/G1 phase accompanied by p21 activation.
Maimets
T
T
Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia. toivo.maimets@ut.ee
Neganova
I
I
Armstrong
L
L
Lako
M
M
D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation; D002478:Cells, Cultured; D019925:Cyclin A / Q000502:physiology; D019927:Cyclin E / Q000502:physiology; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013237:Stereoisomerism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D002087:Butyrates; D019925:Cyclin A; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D007093:Imidazoles; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D010879:Piperazines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C482203:nutlin 1; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2