Ras-inducible immortalized fibroblasts: focus formation without cell cycle deregulation.
The Ras oncogene transforms cultured murine fibroblasts into malignant, focus-forming cells, whose lack of contact inhibition is evidenced by high saturation densities. In order to investigate the reversibility of Ras transformation, as well as the kinetics of Ras-induced changes, cell lines that conditionally express oncogenic Ras were constructed. Both focus formation and increased saturation density were inducible and fully reversible. In exponentially growing cells, oncogenic Ras-expression had no effect on proliferation rates, Erk phosphorylation, or the level of cyclin D1, and Ras-induction did not confer serum-independent growth. As expected, growth to high density in uninduced cells led to quiescence with a low level of cyclin D1 and no active Erk; in this setting, Ras induction prevented full downregulation of cyclin D1 and inactivation of Erk. Our results show that Ras expression to a level sufficient for transformation leads to relatively subtle effects on known downstream targets, and that the focus formation and increased saturation density growth induced by Ras is not a result of growth factor independence.
Jacobsen
Kivin
K
Department of Molecular Cell Biology, University of Copenhagen, DK-1353 Copenhagen, Denmark.
Groth
Anja
A
Willumsen
Berthe M
BM
D016475:3T3 Cells / Q000166:cytology*; D000818:Animals; D002453:Cell Cycle; D002455:Cell Division; D002461:Cell Line, Transformed / Q000166:cytology; D002472:Cell Transformation, Viral / Q000235:genetics*; D003114:Colony-Forming Units Assay; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D019938:Cyclin D1 / Q000096:biosynthesis / Q000502:physiology*; D005814:Genes, Viral; D011905:Genes, ras; D020935:MAP Kinase Signaling System; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D015689:Oncogene Protein p21(ras) / Q000502:physiology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction; D014162:Transfection
D016895:Culture Media, Serum-Free; D011993:Recombinant Fusion Proteins; D019938:Cyclin D1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras)