Partial reversion of the transformed phenotype in HRAS-transfected tumorigenic cells by transfer of a human gene.
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 introduced into FE-8 cells by cotransfection with the plasmid pY3 conferring hygromycin B resistance. Transfectants were selected in medium containing hygromycin B. HRAS-transformed FE-8 cells showed an increased sensitivity toward ouabain when compared to their normal counterparts. Therefore, 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 transfectants in nude mice was reduced but not completely abolished. 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 suppressor 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.
D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000502:physiology; D011905:Genes, ras; D006801:Humans; D007163:Immunosorbent Techniques; D010042:Ouabain / Q000494:pharmacology; D010641:Phenotype; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051381:Rats; D013489:Suppression, Genetic; D014162:Transfection
D011518:Proto-Oncogene Proteins; D010042:Ouabain; D019204:GTP-Binding Proteins