Transformation of diploid human fibroblasts by transfection of N-ras-oncogenes.

Abstract

To determine if diploid human fibroblasts can be transformed by the N-ras oncogene found in human tumors, early passage cell lines were transfected with an N-ras oncogene from human leukemia cell line 8402 cloned into a high expression plasmid (pSV N-ras), with the N-ras oncogene from human fibrosarcoma cell line HT1080 cloned into pNR-MG1, or with pSV2neo as a control. Each plasmid carries the neo gene coding for Geneticin resistance, but in pSV N-ras, the endogenous promoter of the N-ras gene has been eliminated, and the gene has been inserted between the viral long-terminal repeat (LTR) and the neo gene so that transcription initiated from the LTR must transcribe the N-ras gene before transcribing neo. In pNR-MG1, transcription of the N-ras gene is driven from its endogenous promoter, and the neo gene is transcribed from an SV40 viral promoter, as in pSV2neo. When the transfectants were selected for Geneticin resistance, 70% of the colonies formed with pSV N-ras consisted of morphologically altered cells. Less than 5% of the drug resistant colonies formed with pNR-MG1 had cells with altered morphology, and the change in morphology was much less distinct than with pSV N-ras. No colonies of morphologically-transformed cells were found with pSVneo. If the transfected populations were not selected in Geneticin, but were simply allowed to grow to confluence, very distinct foci composed of morphologically-altered cells could be seen against a contact-inhibited monolayer with pSV N-ras. Foci formed by the pNR-MG1 population were subtle and much less distinct. No foci were found with pSV2neo. Representative colonies and foci of morphologically-transformed cells were isolated. Those from pNR-MG1 transfection reverted to a normal morphology after 5-10 population doublings. Most of those from pSV N-ras transfection either reverted or senesced after 5-10 population doublings. However, progeny of two colonies expressed stable morphological transformation throughout a normal life span equal to that of age-matched pSV2neo controls. Both of these stably transformed cell strains exhibited anchorage independence and formed distinct foci. Immunoprecipitation analysis showed that these cells produced much larger amounts of N-ras protein than did pSV2neo-transfected control cells. However, these two cell strains did not exhibit an infinite life span in culture and were unable to form tumors in athymic mice.