High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27(Kip) Cdk-complexes.
The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27(Kip1) levels rose and the inhibitor accumulated in both Cdk4 and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27(Kip1) induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density.
Department of Molecular Cell Biology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen, Denmark. agr@cancer.dk
Anja
A
Groth
Berthe M
BM
Willumsen
D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D003260:Contact Inhibition; D019938:Cyclin D1 / Q000378:metabolism; D056742:Cyclin D2; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
C486196:Ccnd2 protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D056742:Cyclin D2; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D015689:Oncogene Protein p21(ras)