When wild-type mouse embryo cells are stably transfected with a plasmid constitutively overexpressing the epidermal growth factor (EGF) receptor (EGFR), the resulting cells can grow in serum-free medium supplemented solely with EGF. Supplementation with EGF also induces in these cells the transformed phenotype (growth in soft agar). However, when the same EGFR expression plasmid is introduced and overexpressed in cells derived from littermate embryos in which the insulin-like growth factor I (IGF-I) receptor genes have been disrupted by homologous recombination, the resulting cells are unable to grow or to be transformed by the addition of EGF. Reintroduction into these cells (null for the IGF-I receptor) of a wild-type (but not of a mutant) IGF-I receptor restores EGF-mediated growth and transformation. Our results indicate that at least in mouse embryo fibroblasts, the EGFR requires the presence of a functional IGF-I receptor for its mitogenic and transforming activities.

NCT01086826, www.clinicaltrials.gov.

No abstract

Mouse embryo cells expressing a wild-type number of insulin-like growth factor I receptors (IGF-IR) (W cells) can be transformed either by simian virus 40 large T antigen (SV40 T) or by overexpressed insulin receptor substrate 1 (IRS-1), singly transfected. Neither SV40 T antigen nor IRS-1, individually, can transform mouse embryo cells with a targeted disruption of the IGF-IR genes (R ؊ cells). However, cotransfection of SV40 T antigen and IRS-1 does transform R؊ cells. In this study, using different antibodies and different cell lines, we found that SV40 T antigen and IRS-1 are coprecipitated from cell lysates in a specific fashion, regardless of whether the lysates are immunoprecipitated with an antibody to SV40 T antigen or an antibody to IRS-1. The same antibody to SV40 T antigen, however, fails to coprecipitate another substrate of IGF-IR, the transforming protein Shc, and two other signal-transducing molecules, Grb2 and Sos. Finally, an SV40 T antigen lacking the amino-terminal 250 amino acids fails to coprecipitate IRS-1 and also fails to transform R ؊ cells overexpressing mouse IRS-1. These experiments indicate that IRS-1 associates with SV40 T antigen and that this association plays a critical role in the combined ability of these proteins to transform R ؊ cells. This finding is discussed in light of the crucial role of the IGF-IR in the establishment and maintenance of the transformed phenotype.The insulin-like growth factor I (IGF-I) receptor (IGF-IR) (56) plays a crucial role in the establishment and maintenance of the transformed phenotype. Antibodies to IGF-IR (1, 18), antisense expression plasmids to either IGF-I (55), IGF-IR (4, 40, 41, 48), or IGF-II (6), and dominant negative mutants of IGF-IR (25, 37) can all reverse the transformed phenotype and/or inhibit tumorigenesis. Conversely, overexpression of the wild-type (but not a mutant) IGF-IR induces transformation (7,19,25,28,46), while overexpression of IGF-II in transgenic mice increases the incidence of certain malignancies (42).Recently, we have generated from mouse embryos homozygous for a targeted disruption of the IGF-IR genes and from their wild-type littermates (2, 26) cell lines of 3T3-like fibroblasts, designated, respectively, R Ϫ (receptor minus) and W (wild-type) cells (46,47). R Ϫ cells grow in 10% serum (albeit more slowly than W cells) but do not grow at all in serum-free medium supplemented with the growth factors that sustain the growth of W cells, and of other 3T3-like cells, such as plateletderived growth factor (PDGF), epidermal growth factor, IGF-I, IGF-II, insulin at supraphysiological concentrations, and fibroblast growth factors (7, 46). In addition, R Ϫ cells are refractory to transformation by simian virus 40 (SV40) T antigen (47) or by other oncoproteins, such as an activated HaRas (46, 52), singly or in combination. The growth deficits of R Ϫ cells, including their resistance to transformation, are abrogated by the stable transfection of a plasmid expressing a wild-type (but not a mutant) human IGF-IR cDNA (7,9,25...