Abrogation of p27Kip1 by cDNA antisense suppresses quiescence (G0 state) in fibroblasts.
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.
Rivard
N
N
Centre de Biochimie-CNRS, Université de Nice, Parc Valrose, 06108 Nice, France.
L'Allemain
G
G
Bartek
J
J
Pouysségur
J
J
D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000502:physiology; D004247:DNA / Q000096:biosynthesis; D016373:DNA, Antisense / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000378:metabolism; D016193:G1 Phase / Q000235:genetics / Q000502:physiology; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D016192:Resting Phase, Cell Cycle / Q000235:genetics* / Q000502:physiology*; D016196:S Phase / Q000235:genetics / Q000502:physiology; D014162:Transfection; D025521:Tumor Suppressor Proteins
C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016373:DNA, Antisense; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D018844:Cyclin-Dependent Kinases