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Molecular and Cellular Biology, September 2000, p. 6741-6754, Vol. 20, No. 18
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Uncoupling between Phenotypic Senescence and Cell Cycle Arrest in Aging p21-Deficient Fibroblasts

Vjekoslav Dulic',1,* Georges-Edouard Beney,1 Guillaume Frebourg,1 Linda F. Drullinger,2 and Gretchen H. Stein2

Centre de Recherche en Biochimie Macromoléculaire (CRBM)-Centre National de la Recherche Scientifique (CNRS), 34293 Montpellier, France,1 and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-03472

Received 17 December 1999/Returned for modification 7 February 2000/Accepted 8 June 2000

Irreversible G1 arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21Cip1/SDI1/WAF1 and p16Ink4A. 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 G1 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 G1 arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.

* Corresponding author. Mailing address: Centre de Recherche en Biochimie Macromoléculaire (CRBM)-Centre National de la Recherche Scientifique (CNRS), UPR 1086, 1919, Route de Mende, 34293 Montpellier, France. Phone: (33) 4-67613337. Fax: (33) 4-67521559. E-mail: dulic{at}crbm.cnrs-mop.fr.

Molecular and Cellular Biology, September 2000, p. 6741-6754, Vol. 20, No. 18
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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