STEPWISE TRANSFORMATION OF ASTROCYTES BY SIMIAN-VIRUS-40 LARGE T-ANTIGEN AND EPIDERMAL GROWTH-FACTOR RECEPTOR OVEREXPRESSION

We have investigated the transformed phenotype of neonatal mouse corti cal astrocytes immortalized by retrovirus-mediated transfer of the SV4 0 large T antigen gene, Expression of T antigen was driven by the Molo ney murine leukemia virus long terminal repeat, Cell lines were select ed based on coexpression of neomycin resistance, which provides a sele ction method believed to be unbiased for transformation state, Astrocy te cell lines derived in this manner express T antigen over a relative ly narrow range (similar to 4-fold), are contact inhibited, are able t o enter a quiescent state in the presence of growth factors, and do no t readily form colonies in soft agar. Compared to mortal astrocytes, t he population growth rate is increased 3-fold, saturation densities ar e 4-fold higher, and the genome is relatively unstable as measured by the presence of DNA-aneuploid stem lines and by changes in DNA ploidy over time, However, changes in transformation phenotype occur at a low rate, making the cell lines amenable to experimentation. Most often, the growth phenotype remained unchanged during months of culture, Tran sfection of an epidermal growth factor receptor (EGFR) gene was used t o generate a subline that was conditionally transformed (colony format ion in soft agar was dependent on transforming growth factor cu), v-ra f transfection was used to generate constitutive transformation. Thus, these cell lines appear to be excellent experimental models for progr essive transformation. With them, untested hypotheses of brain tumor p rogression derived from human genetic studies may be tested experiment ally.