Abrogation of p27Kip1 by cDNA Antisense Suppresses Quiescence (G0 State) in Fibroblasts

doi:10.1074/jbc.271.31.18337

Abrogation of p27^Kip1 by cDNA Antisense Suppresses Quiescence (G₀ State) in Fibroblasts*

From the Centre de Biochimie-CNRS, Université de Nice, Parc Valrose, 06108 Nice, France and the
^§ Department of Cell Cycle and Cancer, Division for Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark

^‡ Supported by a Canadian NSRC postdoctoral fellowship. To whom correspondence should be addressed. Tel.: 33-92-07-64-27; Fax: 33-92-07-64-32.

Abstract

Progression of eukaryotic cells through the cell cycle is governed by the sequential formation, activation, and subsequent inactivation of a series of cyclin-dependent kinase (Cdk) complexes. p27^Kip1 (p27) is a Cdk inhibitor that blocks, in vitro, the activity of cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2 as well as cyclin A-Cdk2, a complex active during S phase. The level of p27 protein expression, usually high in G₀/G₁ resting cells, declines as cells progress toward S phase and enforced expression of p27 in fibroblasts causes G₁ arrest. This situation prevails in CCL39, a Chinese hamster lung fibroblast cell line (this report). However, in addition to p27, several other Cdk inhibitors known to alter G₁ progression coexist in most mammalian cells. To investigate the specific contribution of p27 in the control of the mitogen-sensitive G₀/G₁ arrest, we specifically reduced its synthesis by expressing a full-length p27 antisense cDNA in CCL39 cells. Interestingly, reduction of up to 90% of p27 protein expression increased both basal and serum-stimulated gene transcription of cyclin D1, cyclin A, dihydrofolate reductase, and DNA synthesis reinitiation. Moreover, overexpression of this antisense allows cells to grow for several generations in a serum-free medium supplemented with insulin and transferrin only, thus suggesting that p27-depleted cells cannot exit the cell cycle. These effects were fully reversed by coexpression of a plasmid encoding p27 sense. We conclude that p27, by setting the level of growth factor requirement, plays a pivotal role in controlling cell cycle exit, a fundamental step in growth control.

Footnotes

↵* This work was supported in part by grants from Centre National de la Recherche Scientifique (CNRS), la Ligue Nationale Contre le Cancer, and l'Association pour la Recherche contre le Cancer (ARC). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Cdk

cyclin-dependent kinase

CKI

Cdk inhibitor

MAP

mitogen-activated protein kinase

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum.
↵² J. Bartek, unpublished data.
↵³ G. L'Allemain, J. N. Lavoie, and J. Pouysségur, manuscript in preparation.
↵⁴ Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouysségur, J. (1996) J. Biol. Chem., in press.
↵⁵ N. Rivard, G. L'Allemain, and J. Pouysségur, unpublished observations.
- Received April 4, 1996.
- Revision received June 5, 1996.