Ras-inducible immortalized fibroblasts: focus formation without cell cycle deregulation

Abstract

The Ras oncogene transforms cultured murine ®broblasts 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 Rasexpression had no e ect on proliferation rates, Erk phosphorylation, or the level of cyclin D1, and Rasinduction 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 su cient for transformation leads to relatively subtle e ects 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.

Induction of p202, a modulator of apoptosis, during oncogenic transformation of NIH 3T3 cells by activated H‐Ras (Q61L) contributes to cell survival

“…Thus, raising the possibility that the increased levels of p202 by antagonizing Ras-induced cell growth stimulation limit deregulation of cell cycle progression. Together, these observations are consistent with the recent report that oncogenic Ras can transform NIH 3T3 cells without deregulation of cell cycle [Jacobsen et al, 2002].…”

“…However, transfection of cells with empty vector did not result in such morphological changes. Consistent with the previous report [Jacobsen et al, 2002], at lower cell densities, cultures derived from vectortransfected cells and Ras transformed cells grew at the comparable cell proliferation rate for the first three days (Fig. 1B).…”

c-Jun NH2-Terminal Kinase 2 Is Required for Ras Transformation Independently of Activator Protein 1

“…1A, middle), and the fact that they do not have any defect in Ras transformation, indicates that the JNK2 isoform of JNK is specifically supporting Ras transformation and that the results reported above are not just due to a possible overall lower level of JNK expression in Jnk2À/À MEFs. Furthermore, because retrovirally transduced H-Ras was expressed in the Jnk2À/À cells as efficiently as in the corresponding wild-type and Jnk1À/À cells, the defect in Ras transformation in Jnk2À/À MEFs was not due to lower Ras levels either, which have previously been shown to influence Ras transformation efficiency (23). Thus, our data clearly points out that selective inactivation of the JNK signaling pathway by preventing the expression of the JNK2 isoform of JNK impairs Ras transformation.…”

Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation

“…In vitro tumorigenicity assay Focus formation assay was carried out as described previously, and NIH3T3 fibroblasts were used as background cells for focus formation assay (Jacobsen et al, 2002;Nielsen et al, 2007).…”