Partial reversion of the transformed phenotype in HRAS-transfected tumorigenic cells by transfer of a human gene (suppressor gene/morphological transformation/anchorage independence/ouabain selection)

The transformed phenotype of rat FE-8 cells transfected by an activated human HRAS gene was suppressed upon fusion with normal cells. An experimental approach was developed to identify and isolate a human gene capable of suppressing the transforming activity of the HRAS oncogene in FE-8 cells. Genomic DNA from human placenta was intro- duced Into FE-8 cells by cotransfection with the plasmid pY3 conferring hygromycin B resistance. Transfectants were se- lected in medium containing hygromycin B. HRAS-trans- formed FE-8 cells showed an increased sensitivity toward ouabain when compared to their normal counterparts. There- fore, the population of transfected hygromycin B-resistant cells was treated with ouabain to eliminate cells with a transformed phenotype. Ouabain selection resulted in a small number of cell clones exhibiting a more normal phenotype. The clones had lost the morphology of transformed cells and required anchorage for growth. The tumorigenicity of trans- fectants in nude mice was reduced but not completely abol- ished. FE-8 revertants continued to express the p21 RAS protein. Human repetitive sequences contained in the DNA of a secondary transfectant were used for isolation of the sup- pressor gene from reverted FE-8 cells. The cloned DNA fragment was transfected into tumorigenic FE-8 cells and conferred a partial reversion of the transformed phenotype.