Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts.
Irreversible G(1) arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21(Cip1/SDI1/WAF1) and p16(Ink4A). To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G(1) arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21(-/-) mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G(1) arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.
Dulić
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V
Centre de Recherche en Biochimie Macromoléculaire (CRBM)-Centre National de la Recherche Scientifique (CNRS), Montpellier, France. dulic@crbm.cnrs-mop.fr
Beney
G E
GE
Frebourg
G
G
Drullinger
L F
LF
Stein
G H
GH
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D002478:Cells, Cultured; D016922:Cellular Senescence; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000502:physiology*; D004791:Enzyme Inhibitors; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016193:G1 Phase; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008938:Mitosis; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C068651:E6 protein, Human papillomavirus type 16; D004791:Enzyme Inhibitors; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases