Abstract
A senescence-like growth arrest is induced in mouse primary embryo fibroblasts by inhibitors of phosphoinositide 3-kinase (PI3K). We observed that senescence-like growth arrest is correlated with an increase in p27(Kip1) but that down-regulation of other cyclin-dependent kinase (CDK) inhibitors, including p15(INK4b), p16(INK4a), p19( INK4d), and p21(Cip1) as well as other negative cell cycle regulators such as p53 and p19(ARF), implies that this senescence-related growth arrest is independent of the activity of p53, p19(ARF), p16(INK4a), and p21(Cip1), which are associated with replicative senescence. The p27(Kip1) binds to the cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We demonstrated that ectopic expression of p27(Kip1) can induce permanent cell cycle arrest and a senescence-like phenotype in wild-type mouse embryo fibroblasts. We also obtained results suggesting that the kinase inhibitors LY294002 and Wortmannin arrest cell growth and induce a senescence-like phenotype, at least partially, through inhibition of PI3K and protein kinase B/Akt, activation of the forkhead protein AFX, and up-regulation of p27(Kip1)expression. In summary, these observations taken together suggest that p27(Kip1) is an important mediator of the permanent cell cycle arrest induced by PI3K inhibitors. Our data suggest that repression of CDK2 activity by p27(Kip1) is required for the PI3K-induced senescence, yet mouse embryo fibroblasts derived from p27(Kip1-/-) mice entered cell cycle arrest after treatment with LY294002. We show that this is due to a compensatory mechanism by which p130 functionally substitutes for the loss of p27(Kip1). This is the first description that p130 may have a role in inhibiting CDK activity during senescence.
Highlights
B Present address: Dept. of Biochemistry, New York University Medical Center, 550 First Ave., New York, NY 10016
We observed that senescencelike growth arrest is correlated with an increase in p27Kip1 but that down-regulation of other cyclindependent kinase (CDK) inhibitors, including p15INK4b, p16INK4a, p19 INK4d, and p21Cip1 as well as other negative cell cycle regulators such as p53 and p19ARF, implies that this senescence-related growth arrest is independent of the activity of p53, p19ARF, p16INK4a, and p21Cip1, which are associated with replicative senescence
LY294002 Arrests Early Passage Mouse Embryo Fibroblasts at G1 Phase of the Cell Cycle—A previous report has described that the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002 induces a senescence-like growth arrest in human diploid fibroblasts [40]
Summary
B Present address: Dept. of Biochemistry, New York University Medical Center, 550 First Ave., New York, NY 10016. LY294002-induced Accumulation of p27Kip1 Is Independent of p16INK4a, p21Cip1, p53, and p19ARF—To better understand this cell cycle arrest-associated accumulation of p27Kip1 after LY294002 treatment, we treated mouse embryo fibroblasts isolated from mice devoid of negative cell cycle regulators previously shown to accumulate during replicative senescence and examined the expression of p27Kip1 using Western blotting after 24 h (Fig. 3C).
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