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Molecular and Cellular Biology, September 2000, p. 6741-6754, Vol. 20, No. 18
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
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
,1,*
/
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.
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