Transformation of rabbit vascular smooth muscle cells by transfection with the early region of SV40 DNA

Abstract

Rabbit aortic smooth muscle cells (SMC) and Rb-1 cells, a continuous line of the same origin, were transformed by transfection with pSV3-neo DNA, a plasmid containing the SV40 early region linked to the neoR resistance gene. Transformed clones were selected in G418-containing medium at a rate of 10(-4) per cell. All transformed clones were immortalized and contained in the early passages two free recombined plasmids derived from pSV3-neo. At advanced passages pSV3-neo sequences were found integrated in the cellular genome. Transformed cells had an altered morphology and growth pattern that differed among clones. Some clones reached high density in low-serum medium. All the clones stained positively for the intranuclear T antigen. Some clones had distinct transcripts for the large T and small t antigens, while in others only larger or truncated transcripts were found. Alpha-actin filaments were visualized by immunofluorescent staining in all the clones, but Northern blot analysis revealed a significant reduction in transcripts for this actin. All the transformed clones accumulated, to a variable extent, cholesteryl esters after incubation with beta very low-density lipoprotein. Six of the eight transformed clones maintained a diploid chromosome number, but there was an increase in structural chromosome aberrations, predominantly dicentrics. Transfection of pSV3-neo into rabbit vascular SMCs is an efficient model for obtaining transformed clonal populations. These clones show some phenotypic changes that may be relevant to the study of atherogenesis.