Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay.

We have isolated, cultured, and immortalised three new BigBlue transgenic rat cell lines for the study of mutation induction in vitro. The two epithelial cell lines, from the mammary gland and oral cavity, were designated BBR/ME and BBR/OE, respectively, and the third is a mammary fibroblast line designated BBR/MFib. We have characterised these cell lines with respect to chromosome number and the expression of some cell-specific antigens. The clonogenic survival and cII transgene mutation induction responses of these three cell lines to N-ethyl-N-nitrosourea (ENU) treatment were determined. Both epithelial cell lines were much more sensitive to ENU toxicity than was the fibroblast cell line. However, all cell lines showed similar ENU dose-dependent increases in mutant frequency. We hope that cell lines such as these will extend the power of the BigBlue assay to in vitro studies.

Insulin-like growth factor-I (IGF-I)-dependent activation of pp42/44 mitogen-activated protein kinase occurs independently of IGF-I receptor kinase activation and IRS-1 tyrosine phosphorylation.

The proliferation and metabolism of H4IIE hepatoma cells is apparently mediated through the insulin receptor. These cells, however, also have high-affinity binding sites for insulin-like growth factor-I (IGF-I). Addition of insulin to H4IIE cells increased RNA synthesis, DNA synthesis and cell number. IGF-I, on the other hand, was ineffective at concentrations equivalent to the lowest effective insulin dose, although stimulation was observed with concentrations 100-fold higher. Similar results were obtained when glucose uptake was measured. Western blot analysis demonstrated that tyrosine phosphorylation patterns produced by insulin and IGF-I differed. In particular, phosphorylation of insulin receptor substrate-1 (IRS-1) was evident after treatment with insulin, but not after treatment with IGF-I. Correspondingly, insulin, but not IGF-I, stimulated receptor tyrosine kinase activity. In contrast with these results, both insulin and IGF-I induced mitogen-activated protein (MAP) kinase phosphorylation and activity at a concentration of 10 nM. The correlation between insulin-dependent and IGF-I-dependent MAP kinase activation was confirmed by Western blot analysis of phosphorylated MAP kinase kinase (MEK). These results suggest that phosphorylation of IRS-1 is essential for both cell proliferation and glucose metabolism, but is uncoupled from the MAP kinase cascade. Furthermore, stimulation of MEK and MAP kinase is independent of receptor tyrosine kinase activity.