Morphological transformation, focus formation, and anchorage independence induced in diploid human fibroblasts by expression of a transfected H-ras oncogene.
Morphological transformation, focus formation, and anchorage independence induced in diploid human fibroblasts by expression of a transfected H-ras oncogene.
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发表时间：
1987-11
期刊：
影响因子：
11.2
通讯作者：
Peter J. Hurlin;Dennis G. Fry;Veronica M. Maher;J. Mccormick
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文献类型：
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作者：
Peter J. Hurlin;Dennis G. Fry;Veronica M. Maher;J. Mccormick
为了确定正常人成纤维细胞如何响应 T24 H-ras 癌基因的高表达，我们用质粒载体 pHO6T1 (D. A. Spandidos 和 N. M. Wilkie, Nature (Lond.), 310:469-475, 1984)转染此类细胞，该载体含有具有 5' 和 3' 增强子序列的 T24 H-ras 癌基因和氨基糖苷类。磷酸转移酶基因赋予对药物 G418 的抗性。转染和筛选后获得的G418抗性集落中，约1.5%由表现出明显形态转变的细胞组成；即，它们比正常成纤维细胞具有更高的折射性并且更圆。 DNA杂交分析显示形态转化的细胞含有转染的T24 H-ras癌基因，放射免疫沉淀分析显示它们表达T24 H-ras蛋白产物M,21,000蛋白。形态学转化的细胞在软琼脂中形成集落的频率比用含有正常细胞H-ras基因的对照质粒转染的细胞高至少60倍。还可以通过转染后在非选择性培养基中生长至汇合时形成不同病灶的能力来鉴定用质粒 pHO6T1 转染的细胞。这项研究表明，培养中的正常二倍体人类成纤维细胞可以通过 H-ras 癌基因转染进行转化，并且这种转化与突变型 Mr 21,000 蛋白的表达相关。
In an attempt to determine how normal human fibroblasts respond to high expression of the T24 H-ras oncogene, we tranfected such cells with the plasmid vector pHO6T1 (D. A. Spandidos and N. M. Wilkie, Nature (Lond.), 310:469-475, 1984), containing the T24 H-ras oncogene with 5' and 3' enhancer sequences, and the aminoglycoside phosphotransferase gene which confers resistance to the drug, G418. Approximately 1.5% of the G418-resistant colonies obtained after transfection and selection consisted of cells exhibiting obvious morphological transformation; i.e., they were highly refractile and more rounded than normal fibroblasts. DNA hybridization analysis showed that the morphologically transformed cells contained the transfected T24 H-ras oncogene, and radioimmunoprecipitation analysis showed that they were expressing the T24 H-ras protein product, M, 21,000 protein. Morphologically transformed cells formed colonies in soft agar at a frequency at least 60 times higher than that of cells that had been transfected with the control plasmid containing the normal cellular H-ras gene. Cells transfected with plasmid pHO6T1 could also be identified by their ability to form distinct foci when grown to confluence in nonselective medium following transfection. This study demonstrates that normal diploid human fibroblasts in culture can be transformed by transfection with a H-ras oncogene, and that such transformation correlates with expression of the mutant Mr 21,000 protein.