Schaefer R, Iyer J, Iten E, Nirkko A C
Ludwig Institute for Cancer Research, Inselspital, Bern, Switzerland.
Proc Natl Acad Sci U S A. 1988 Mar;85(5):1590-4. doi: 10.1073/pnas.85.5.1590.
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.
用活化的人HRAS基因转染的大鼠FE - 8细胞的转化表型在与正常细胞融合后受到抑制。开发了一种实验方法来鉴定和分离能够抑制FE - 8细胞中HRAS癌基因转化活性的人类基因。通过与赋予潮霉素B抗性的质粒pY3共转染，将人胎盘基因组DNA导入FE - 8细胞。在含有潮霉素B的培养基中选择转染子。与正常的FE - 8细胞相比，HRAS转化的FE - 8细胞对哇巴因表现出更高的敏感性。因此，用哇巴因处理转染的潮霉素B抗性细胞群体，以消除具有转化表型的细胞。哇巴因选择产生了少数表现出更正常表型的细胞克隆。这些克隆失去了转化细胞的形态，并且生长需要贴壁。转染子在裸鼠中的致瘤性降低但并未完全消除。FE - 8回复体继续表达p21 RAS蛋白。二次转染子DNA中含有的人类重复序列被用于从回复的FE - 8细胞中分离抑制基因。将克隆的DNA片段转染到致瘤性FE - 8细胞中，使转化表型部分回复。
