Characterization of human vascular smooth muscle cells transformed by the early genetic region of SV40 virus.

Human arterial smooth muscle cells transfected with the plasmid pSV3-neo, which contains the SV40 virus early region and the neor gene, developed colonies of morphologically transformed cells. Five cell strains were initiated from these colonies and could be subcultivated for up to 9 months before entering a stage of crisis that ended their life span. Deoxyribonucleic acid (DNA) molecules containing viral sequences were found free and integrated in the transformed cells. The intranuclear SV40 large T antigen and the p53 cellular protein were expressed in the transformed cells. Most of the transformed cells were spindle shaped but some were large and multinucleated. The modal chromosome numbers were in the triploid range, and aberrations, particularly dicentrics, were common. The transcripts for smooth muscle actins were significantly reduced and there were less alpha-actin filaments detected by immunofluorescence. Cytochemical staining disclosed a large accumulation of lipid droplets in the transformed cells incubated with rabbit hypercholesterolemic beta-very-low-density lipoprotein. Chemical analysis showed that cholesteryl esters were significantly elevated in these cells. Phenotypic changes induced in human vascular smooth muscle cells by SV40 early genes are similar to those found in smooth muscle cells from atherosclerotic lesions and may indicate common pathogenetic mechanisms.

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

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.

Immortalization of rabbit vascular smooth muscle cells after transfection with a fragment of the BglII N region of herpes simplex virus type 2 DNA.

Rabbit arterial smooth muscle cells were transfected with the pBC24neo plasmid DNA which consists of a sequence of 790 base pairs from the BglII N fragment of herpes simplex virus type 2 DNA linked to the neo resistance gene. Selection in G418-containing medium resulted in eleven immortalized clones which showed increased growth rate and saturation densities. One of the eleven clones maintained the transforming DNA sequence integrated in the cellular genome and showed continuous resistance to G418, but Southern blot analysis of the other ten immortalized clones did not detect pBC24neo DNA sequences. Possible mechanisms of herpes simplex virus induced immortalization and their implications in the development of atheromatous plaques are discussed.

Characterization of a continuous smooth muscle cell line derived from rabbit aorta.

A spontaneously arising continuous cell line (Rb-1) derived from collagenase-elastase digested rabbit aorta has been propagated in vitro for over 100 passages. During this period, the Rb-1 cells remained spindle-shaped and formed regularly oriented parallel bundles. After Passage 50, Rb-1 cells were found to be serum-independent in their growth and reached higher saturation density than rabbit aorta smooth muscle cells. Alpha-actin and desmin filaments were detected by immunostaining in Rb-1 cells and early passage of rabbit aorta smooth muscle cells. The proportion of alpha-actin transcripts in Rb-1 cells was lower than that of transcripts for beta- and gamma-actins. The modal chromosome number was maintained at 44 between Passages 11 and 60, and two marker chromosomes were constantly present. Infection of Rb-1 cells with two strains of herpes simplex virus type 1 resulted in high titers of virus, whereas a herpes simplex virus type 2 temperature-sensitive mutant replicated only at the permissive temperature. The Rb-1 cell line could be used for the study of vascular smooth muscle cell proliferation and their interaction with viruses.

Chromosome changes in rat embryo cell lines transformed by temperature-sensitive mutants and sheared DNA of herpes simplex virus.

The chromosomes of six rat embryo cell lines transformed with herpes simplex virus (HSV) temperature-sensitive (ts) mutants were examined at different passages of in vitro cultivation. Two cell lines were predominantly diploid, one cell line was hyperdiploid, one cell line was pseudodiploid, and two cell lines were hypotetraploid. In near-diploid cell lines chromosome No. 9 was most frequently involved in chromosome changes. All three cell lines derived from tumors obtained after one transplantation of HSV-transformed cells into baby rats were pseudodiploid, but each had different marker chromosomes. Chromosome No. 15 was involved in the formation of two out of four marker chromosomes. Four cell lines derived from tumors developing after two and three transplantations were hypodiploid and showed large chromosome variation. The occurrence of 25 marker chromosomes in three tumor-derived cell lines resulted in gains in parts from chromosomes No. 2, 6, and 7. One marker chromosome had a homogeneously faintly stained region. Chromosomes No. 2, 3, 7, and 12 were more frequently involved in the formation of marker chromosomes. No chromosome change was found to be specifically associated with HSV-induced transformation of rat cells, but chromosome changes in tumor-derived cell lines may provide selective advantage for survival and autonomous growth in the host animal.

[Production and characterization of some mouse embryo cellular substrates in vitro]. / Obtínerea si caracterizarea unor substrate celulare embrionare de Soarece in vitro

Cell cultures with a different multiplication potential in vitro, depending upon the strain source used, were obtained from mouse embryos belonging to the CVA, C57 Black A2G and Swiss strains. Only the Swiss 12 culture underwent spontaneous transformation and was carried through more than 50 passages in vitro. The Swiss-12 substrate proved not to be contaminated either by viruses or micoplasma. It is less sensitive than other elective cell substrates to infection with attenuated polioviruses, cytopathogenic Coxsackie A9 virus and vaccinia virus, but its sensitivity to infection with Herpes simplex type 1 virus is similar to that of human embryo fibroblasts. After a high number of passages the Swiss-12 substrate permits, in comparison to other cell substrates (human heteroploid Hep-2 line, human embryo fibroblasts), a highly efficient qualitative differentiation between the growth media and calf serum.