In an attempt to determine how normal human fibroblasts respond to high expression of the T24 H-ras oncogene, we transfected 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, Mr 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.
This research was supported in part by Department of Energy Grant DE-FG02-87-ER60524 and by Department of Health and Human Services Grant CA21289 from the National Cancer Institute. D. G. F. is a recipient of a Leukemia Society of America Special Fellowship Award.