Growth in serum‐free medium of NIH3T3 cells transformed by the EJ‐H‐<i>ras</i> oncogene: Evidence for multiple autocrine growth factors

Pironin, Martine; Clément, Gilles; Benzakour, Omar; Barritault, Denis; Lawrence, David A.; Vigier, Philippe

doi:10.1002/ijc.2910510624

Cited by 18 publications

(12 citation statements)

References 31 publications

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“…The cyclin D1, fos and other immediate early/delayed early genes are Ras-inducible (SchoÈ nthal et al, 1988;Albanese et al, 1995), and cdk4-cyclin D1 kinase (Villalonga et al, 2000) and the E2F transcription factors (Fan and Bertino, 1997;Gjoerup et al, 1998) are active in Rastransformed, but not parental, serum starved cells. This ®nding is in accord with many reports (Kaplan et al, 1982;Durkin and Whit®eld, 1986;Zhan and Goldfarb, 1986;Pironin et al, 1992;Winston et al, 1996;Villalonga et al, 2000), the results of which argue that activated Ras can induce DNA synthesis, but that cell division in the absence of serum may require Rasinduced autocrine growth-factor production.…”

Section: Introductionsupporting

confidence: 91%

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

2002

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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.

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Section: Introductionsupporting

confidence: 91%

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

2002

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“…After 48 h, cell extracts were prepared and a Western analysis for MHC was performed as described in Figure 1 mutually exclusive events (Visscher and Heppner, 1996). Oncogenic mutation of ras genes has been implicated as a causative event throughout the process of tumorigenesis (Weyman and Stacey, 1996) and expression of oncogenic Ras is correlated with the secretion of both growth and angiogenic factors (Pironin et al, 1992;Dawson et al, 1995;Rak et al, 1995;Berkowitz et al, 1996). While the signi®cance of autocrine growth factor loops to the proliferation of tumor cells is accepted dogma, the contribution of autocrine growth loops to the inhibition of differentiation has not been extensively explored.…”

Section: Discussionmentioning

confidence: 99%

“…Autocrine secretion of growth factors assists tumor cells in escaping normal growth constraints (Sporn and Roberts, (1985). Expression of oncogenic Ras correlates with the secretion of both growth (Pironin et al, 1992;Dawson et al, 1995;Berkowitz et al, 1996) and angiogenic factors (Rak et al, 1995). The mammalian ras family consists of three genes, Ha-, K-and N-, which each encode 21 kD membrane tethered proteins (Lowy and Willumsen, 1993).…”

Section: Introductionmentioning

confidence: 99%

Oncogenic Ras-induced secretion of a novel inhibitor of skeletal myoblast differentiation

Weyman

Wolfman

1997

Oncogene

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Expression of oncogenic H-Ras in 23A2 myoblasts (A2:H-Ras cells) is su cient to induce both a transformed phenotype and a di erentiation-defective phenotype. Because oncogenic Ras is known to induce the secretion of several di erent growth factors involved in maintaining the transformed phenotype of both ®broblast and epithelial cells, we explored the possibility that expression of oncogenic Ras in 23A2 myoblasts might lead to the secretion of a factor which inhibits di erentiation. The di erentiation of 23A2 myoblasts was inhibited (i) by coculture with an equal number of A2:H-Ras cells, (ii) by culture with an equal number of A2:H-Ras cells in the same tissue culture medium on an insert which allowed equilibration of molecules smaller than 1 micron, and (iii) by culture in media previously conditioned by A2:H-Ras cells. Similar results were obtained when 23A2 myoblasts expressing oncogenic NRas were substituted for A2:H-Ras cells in each assay. No inhibition of di erentiation was observed, however, when di erentiation-defective E1A-expressing 23A2 cells or C3H10T1/2 ®broblasts were substituted for A2:HRas cells. The di erentiation inhibitor(s) in media conditioned by A2:H-Ras cells is heat stable, larger than 3 kD, and sensitive to the non-speci®c growth factor antagonist, suramin. Western analyses failed to detect either FGF-2 or TGFb (the known inhibitors of myoblast di erentiation) in media conditioned by A2:H-Ras cells. Furthermore, while FGF-2 is a potent activator of MAP kinase and TGFb is a potent inhibitor of mink lung epithelial cell (CCL64) growth, conditioned media from A2:H-Ras cells does not activate MAP kinase and does not inhibit the growth of CCL64 cells. These results indicate that expression of oncogenic Ras induces the secretion of a novel inhibitor of skeletal myoblast di erentiation. Furthermore, these results are the ®rst to implicate an autocrine/paracrine mechanism in the inhibition of di erentiation by oncogenic Ras.

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“…Suramin inhibits the serum-independent proliferation of Ha-ras but not N-ras transformed ®broblasts Ras-induced cellular transformation, in many instances, is associated with a diminished requirement for exogenous growth factors or serum (Pironin et al, 1992). This Ras-dependent serum-independent proliferation can possibly be attributed to the production of autocrine growth factors, including HB-EGF (McCarthy et al, 1995), TGFa (Berkowitz et al, 1996), VEGF (Rak et al, 1995), IL-1a and IL-6 (Castelli et al, 1994), and IGF-1 (Dawson et al, 1995).…”

Section: Expression Of Activated Ras In C3h10t1/2 ®Broblastsmentioning

confidence: 99%

Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo

Hamilton

Wolfman

1998

Oncogene

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The Ras GTPases function as molecular switches, regulating a multiplicity of biological events. However the contribution, if any, of a speci®c c-Ras isoform (Ha-, N-, or Ki-ras A or B) in the regulation of a given biological or biochemical process, is unknown. Murine C3H10T1/2 ®broblasts transformed with activated (G12V)Ha-ras or (Q61K)N-ras proliferate in serum-free media and have constitutive MAPK activity. The growth factor antagonist, suramin, inhibited the serum-independent proliferation of Ha-ras transformed ®broblasts, but not the serum-independent proliferation of N-ras transformed cells. The inhibition of cell proliferation was concomitant with the abrogation of the constitutive MAPK activity in the Ha-ras transformed ®broblasts. Analysis of the Ras-signalling complexes in immunoprecipitates from Ha-ras transformed cells revealed that Raf-1 co-immunoprecipitated with endogenous c-N-ras but not (G12V)Ha-ras. Pretreatment with suramin resulted in the loss of Raf-1 from c-N-ras immunoprecipitates. A c-N-ras antisense oligonucleotide, which down-regulated c-N-ras protein levels, abrogated the constitutive MAPK activity and serum-independent proliferation of (G12V)Ha-ras transformed cells. The data suggest that Raf-1 has a higher a nity for N-ras then Ha-ras in vivo, and c-N-ras function is required for the serum-independent proliferation of Ha-ras transformed cells.

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Growth in serum‐free medium of NIH3T3 cells transformed by the EJ‐H‐ras oncogene: Evidence for multiple autocrine growth factors

Cited by 18 publications

References 31 publications

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

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

Oncogenic Ras-induced secretion of a novel inhibitor of skeletal myoblast differentiation

Ha-ras and N-ras regulate MAPK activity by distinct mechanisms in vivo

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