American Journal of Pathology, Vol. 139, No. 3, September 1991 Copyright ©) American Association of Pathologists

Characterization of Human Vascular Smooth Muscle Cells Transformed by the Early Genetic Region of SV40 Virus Alain Legrand, Phillip Greenspan, Madan L. Nagpal, Sidonia A. Nachtigal, and Maurice Nachtigal From the Department of Patholog)', University of South Carolina School ofAMedicine, Columbia, South Carolina

Human arterial smooth muscle cells transfected with the plasmid pSV3-neo, which contains the SV40 virus earlv 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 multinucleatedc 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 a-actin filaments detected by immunofluorescence. Cytochemical staining disclosed a large accumulation of lipid droplets in the transformed cells incubated with rabbit hypercholesterolemic 3-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. (AmJPathol 1991, 139:629-640)

Smooth muscle cells (SMCs) are an important component in human and experimental atherosclerotic lesions.1-7 Although the abnormal proliferation of SMCs is known to be a key event in the formation of atheromatous plaques, the stimuli responsible for this pathological process are still hypothetical.>14 The finding that most ather-

omatous plaques are monotypic15'16 was interpreted as evidence that these lesions are similar to benign monoclonal neoplastic growths developing through the clonal proliferation of vascular SMCs15 initiated by a mutation introduced in the genome by tumorigenic and mutagenic agents.17 Experimental evidence supporting this hypothesis was brought by the production of atherosclerotic lesions in chickens treated with chemical carcinogens,6'8'9 and by isolation of transforming sequences from human coronary artery plaque DNA.20 The development of atherosclerosis in chickens infected with the oncogenic Marek's disease virus,2' and the demonstration of sequences related to this avian herpesvirus in the DNA extracted from arteries and embryos of quails susceptible to diet-induced atherosclerosis,22 suggested that viruses may play a role in the cause of this disease. Antigens and nucleic acids of herpes simplex viruses (HSV)23'24 and cytomegalovirus (CMV)25'26 were found in human atherosclerotic arteries, and the frequency of coronary artery disease was increased in the transplanted hearts of recipients with CMV infection.27'26 These findings suggested that viral infection may initiate atherosclerosis by different mechanisms, including transformation of arterial SMCs and alteration of their lipid metabolism.'2930 Our investigation tested the response of human vascular SMCs to the introduction and expression of a transforming viral genetic region. The transforming agent used in our work was the early genetic region of the simian virus 40 (SV40), a virus latent in monkeys and tumorigenic in rodents but not in humans.31 Introduction of SV40 deoxyribonucleic acid (DNA) into different types of human cells, including embryo fibroblasts,32'33 endothelial cells,34 and skeletal muscle,-356 results in complex alterations defined as 'transformation.' The phenotype of transformed cells involves changes in the growth pattern, the cell surface, and the intracellular population of macSupported by grant HL 37052 and HL 43794 from the Department of Health and Human Services, National Heart, Lung and Blood Institute. Accepted for publication May 3, 1991. Address reprint requests to Dr. Maurice Nachtigal, Department of Pathology, University of South Carolina School of Medicine, Columbia, SC 29208.

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romolecules. Simian-virus-40-transformed human diploid fibroblasts have an extended life span and outlive their parental normal cells, but eventually enter a stage called 'crisis,' out of which only exceptional continuous cell lines would arise.37 During 'crisis' the cell number remains constant or declines because successful cell division is balanced or exceeded by cell death.38 Cells in crisis are invariably large and marked by reduced proliferative capacity, loss of attachments to the growth surface, abnormal mitotic figures, and formation of giant multinucleated cells.39 We report here that transfection of human smooth muscle from umbilical cord arteries with a plasmid vector expressing the SV40 early genetic region was followed by the development of transformed cell strains that synthesize the SV40 large T antigen, undergo a phase of active growth, and acquire an extended life span that ends in a 'crisis.' In addition to enhanced proliferation, these cells display intracellular accumulation of cholesteryl esters when exposed to atherogenic lipoproteins and have a considerable reduction in the expression of specific smooth muscle actins. The similarity between these phenotypic changes and those found in cells from atherosclerotic lesions suggests the intervention of common pathogenetic mechanisms.

EcoRl-Pvull fragment of pBR322. This gene encodes for the bacterial phosphotransferase APH (3')1l, which inactivates the aminoglycoside G418.42 This allows for the positive selection of cells expressing this gene by the antibiotic G418. The complete SV40 early genetic region, which encodes the large T and the small t antigens, has been inserted into the unique BamHl site of this plasmid. Plasmid 91 (p91)43 was used in assays for the expression of actin genes.

Transfections Supercoiled pSV3-neo DNA (5 ,ug per 25-cm2 flask) was transfected without carrier DNA by the phosphate calcium precipitate technique44 onto semiconfluent cultures of human arterial SMCs. A glycerol shock (25% glycerol in culture medium) was performed for 1 minute, 4 hours after transfection. Two days later, G418 (200 ,ug/ml) (Gibco Laboratories, Grand Island, NY) was added to the culture medium. Individual clones were pipetted off, transferred, and grown as individual strains in medium containing G418 (200 p.g/ml). Alternatively, cell cultures were maintained and subcultivated without G418 after transfection.

Materials and Methods

Focus Formation Assay

Cell Cultures Umbilical cord arteries were isolated under sterile conditions from term delivered human placentas and primary cultures were prepared from the artery media as described.40'41 The 1-mm2 explants were attached to 25cm2 plastic flasks and covered with NCTC 109 medium supplemented with 10% newborn calf serum, 10% donor horse serum, and L-glutamine (4 mmol/l [millimolar]). The cell cultures originating from individual umbilical cords were maintained separated and were assigned code numbers. As soon as a sufficient number of cells were available, usually at the third passage, each cell population was tested by indirect immunofluorescence for the presence of specific smooth muscle a-actin filaments. Only cultures with 100% cells positive for a-actin smooth muscle actin filaments were used for transfections.

Plasmid DNA The plasmid pSV3-neo,42 obtained from the American Type Culture Collection (Rockville, MD) contains the neo resistance gene (neor) from the transposon Tn5 included in a SV40 transcription unit and inserted in the large

Transformed and normal cell suspensions were prepared in NCTC 109 culture medium supplemented with 2% or 20% fetal calf serum and were seeded in 60-mm petri dishes at a density of 103 cells per dish and incubated at 370C. After 15 days the cells were fixed with methanol and stained with Giemsa solution.

Southern Analysis Cellular DNA was prepared and analyzed following standard techniques.45 Transfers were performed on nylon filters (Amersham Corporation, Arlington Heights, IL) and the probe, pSV3-neo DNA, was labeled with the random priming technique46 to a specificity of 2 x 109 cpm/,ug. Hybridizations were performed 24 hours at 42°C in 50% formamide, 5 x SSPE (1 SSPE = 0.2 mol/l [molar] NaCI, 10 mmol/I sodium phosphate pH 6.8, 1 mmol/l ethylenediaminetetra-acetic acid), 2 x Denhardt's solution (0.2% polyvinyl pyrrolidone, 0.2% bovine serum albumin, 0.2% ficoll), 100 ,ug/ml denatured salmon sperm DNA, 0.5% sodium dodecyl sulfate (SDS), 10% dextran sulfate, and washed in high-stringency conditions (0.1 SSPE) at 650C.

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Northern Analysis Total cellular RNA was isolated by guanidine isothiocyanate extraction and CsCI gradient fractionation by the method of Chirgwin et al47 and separated on 1.5% agarose formaldehyde gel.48 After electrophoresis, transfer to HyBond-N membrane (Amersham Corporation, Arlington Heights, IL) was carried out using the standard technique.45 After baking at 800C for 2 hours, prehybridization of the membrane was performed in 5 x SSPE, 50% formamide, 0.5% SDS, 5 x Denhardt's solution, and 200 ,g/ ml denatured salmon sperm DNA at 42°C. Hybridization was carried out under the same conditions in the presence of 10% dextran sulfate, and 2 x 106 cpm of radiolabeled probe per milliliter hybridization buffer. The probe was prepared according to the standard multiprime DNA labeling protocol, as recommended by the manufacturer (Amersham Corporation, Arlington Heights, IL). Whole SV40 DNA linearized with BamHl (5.3 kb) and the SV40 early genetic region (2.9 kb) from pSV3-neo were used as probes for the detection of transcripts of SV40 T antigen. The Pstl-Pstl fragment (1.1 kb) from p91 was used to detect a-, P-, and y-actin transcripts. The blots were washed under stringent conditions (65C, 0.2 x SSC) and autoradiography was carried out using an intensifying screen at - 700C. A densitometer scan was obtained with a Quick Scan R&D (Helena Laboratories, Beaumont, TX) attached to an Apple 11 Plus computer.

Immunostaining Tests The SV40 large T antigen was identified with the monoclonal antibody PAb9O1. Cells grown on glass coverslips were gently washed with prewarmed phosphatebuffered saline (PBS) and fixed with cold absolute methFigure 1. Morphology of pSV3-neo transformed human arterial SMCs. Cell culture of SVH-1 cell strain at passage 14 shows most cells small and spindle-shaped and few cells large and sometimes multinucleated Giemsa stain, x360.

anol for 20 minutes at room temperature. The fixative was discarded, the cell monolayer was washed thoroughly with PBS, and then covered with antiserum (diluted 1:100) and incubated for 45 minutes at 370C. The cells were washed 3 times with PBS, covered with fluorescein isothiocyanate (FITC)-conjugated affinity isolated goat anti-mouse gamma G immunoglobulin (IgG) (whole molecule, adsorbed with human serum proteins) (Sigma Chemical Co., St. Louis, MO) diluted 1:64 and incubated for 30 minutes at room temperature. The FITCconjugated antibody was discarded, and the cells were repeatedly washed with PBS and mounted in glycerolPBS (3:1, vol/vol). The coverslips were examined with a Universal Zeiss microscope. Alternatively cells were stained for SV40 T antigen by the peroxidaseantiperoxidase method, using as a primary antibody a rabbit polyclonal antibody against SV40 T antigen. The link antibody was biotinylated goat anti-rabbit immunoglobulins. The labeling reagent was peroxidase-labeled avidin. The substrate chromogen was 3-amino-9ethylcarbazole and the counterstain was Harris hematoxylin. Immunostaining of the p53 cellular protein was performed using the p53 murine monoclonal antibody PAbl 22.4 The cells were fixed with cold (- 200C) absolute methanol and incubated for 1 hour at 370C with culture medium from clone 122 hybridoma (American Type Culture Collection). The secondary antibody was FITCconjugated affinity isolated goat anti-mouse IgG (whole molecule, adsorbed with human serum proteins) (Sigma Chemical Co.), diluted 1:64, and incubated for 30 minutes at room temperature. For the immunofluorescent staining of smooth muscle a-actin, the cells were first incubated with anti-a-smooth muscle actin monoclonal antibody50 (Sigma Chemical Co.) diluted 1:400 for 45 minutes at 370C. The secondary antibody was FITC-conjugated goat anti-mouse IgG. Ap-

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Chromosome preparations were obtained by a standard procedure previously described.51 Briefly, actively growing cell cultures were treated with Colcemid (Gibco Laboratories, Grand Island, NY) at final concentration of 0.1 ,ug/ml for 3 hours. The cells were detached with trypsinversene solution and suspended for 30 minutes in a 3:1 hypotonic solution. The cells were centrifuged gently and fixed with cold fixative solution consisting of absolute methanol and glacial acetic acid at a 3:1 ratio (vol/vol). Slides were stained with a 5% Giemsa solution and used for chromosome counting and recording structural chromosome aberrations.

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Lipid Analysis Cells were plated in T-25 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and incubated for 24 hours at 370C. The culture medium was changed to DMEM supplemented with lipoprotein-deficient serum (LPDS) containing 2 mg/ml protein and was incubated for another 24 hours. Cells then were incubated with the same medium or with medium containing 50 pg/ml protein ,-very-low-density lipoprotein (,-VLDL, d < 1.006 g/ml fraction) obtained from rabbits fed a 1% cholesterol diet for 1 to 2 months,52 or purchased from Biomedical Technologies, Inc. Stoughton, Massachusetts. After a 48-hour incubation with the lipoprotein, cells were washed, trypsinized, and extracted into chloroform as described previously.53 Lipids were quantified by fluorescence scanning of Nilered-stained thin-layer chromatography plates.5' Results of the chemical analysis were compared with cellular staining for lipids by the Oil red 0 method and by the Nile red procedure.55 Fluorescent analysis of lipid loading was performed by analysis of cells stained with the Nile red.56 For flow cytofluorometry, cells were suspended in PBS, fixed with 3.7% formaldehyde, and stained with Nile red (100 ng/

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Figure 2. Detection ofpSV3-neo sequences. A: Cellular DNA (10 pg)from five subclones derivedffrom the SVH-1 cell strain (lanes 1 to 5) was cleaved with XbaI and analyzed ey Southern blot with a pSV3-neo probe. B: Cellular DNA (10 g) firom the SVH-2 cell strain was cleaved with Xbal (lane 1) or BamHI (lane 2) and hybridized with pSV3-neo. The size marker is lambda phage DNA cleaved with Hind!!!.

propriate negative and positive controls were introduced for each staining. In some instances the cells were double immunostained for SV40 large T antigen and a-smooth muscle actin by incubating the cells with antia-smooth muscle actin and anti-T antigen monoclonal antibodies. B

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Figure 3. Detection of SV40 transcripts. Total cellular RNA from SVH-1 cells was probed with: (A) 32P-labeled SV40 DNA linearized with BamHI; (B) the 2.9 kb BamHIfragment from pSV3-neo which contains the SV40 early region. In both (A) and (B) the lanes at the left were exposed to x-ray film for 6 hours at - 70'C using two intensifying screens, whereas the lanes at the right were exposedfor 24 hours under identical conditions. In lane 1 is RNA from umbilical cord artery SMCs. In lane 2 isRNA extractedfrom SVH-1 cells. Each lane was loaded with 10 p.g RIVA.

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Figure 4. Immunostaining for the SV40 T antigen. Nuclei from SVH-1 cells at passage 18 are stainedfor the SV40 T antigen with the monoclonal antibody PAb9O1. Polylobulated nuclei and micronuclei are also stained by this antibodv.

ml). Nile red-stained cells, or unstained cells, were analyzed with a Coulter EPICS 753 flow cytometer with the excitation laser set at 488 nm. Intracellular lipid loading was preferentially detected by determining the cellular fluorescence at 515 to 560 nm. Analysis of lipid accumulation of individual cells stained with Nile red was performed using digital imaging fluorescence microscopy.57 This analysis was carried out on an Anchored Cell Analysis Station (ACAS 570) (Meridian Instruments Inc., Okemos, Ml). The ACAS 570 interactive laser cytometer is equipped with a 5-W argon ion laser tuned to 488 nm. The laser power was adjusted at 100 nW. The cells were scanned by the movement of the specimen with a speed of 1.5 mm/second and a step size of 2 . relative to the laser. The scan strength was adjusted to 15% through an acousto-optic modulator. An integrated photomultiplier and a microcomputer provided high-speed data acquisition and transformed fluorescent emissions from points of raster scans of the cells Figure 5. Immunostaining for the p53 cellular protein. Nuclei of SVH- 1 cells at passage 18 were stained with the PAbJ22 monoclonal antibody. Giant polylobulated nuclei and micronuclei show intense staining for this cellular protein.

into two-dimensional pseudocolor images. The fluorescence was translated into 15 pseudocolors assigned across the cell in accordance with its intensity pattern. Data processing was performed on a DASY 9000 workstation. For each treatment, 20 fields of approximately 20 cells per field were scanned by the interactive laser cytometer. The intensity of fluorescence light of each scanned cell was expressed numerically in terms of fluorescence units. Using fluorescence units, populations of cells were represented in bar histograms showing number of scanned cells and fluorescence intensity of each cell.

Results Isolation of pSV3-neo-transformed Human Vascular Smooth Muscle Cells Foci of morphologically transformed cells appeared in cultures of human SMCs about 2 months after transfec-

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tion with pSV3-neo DNA. These foci consisted of cells that were refractile, smaller in size, and showed numerous mitotic figures. Five cell strains58 designated SVH-1, SVH-2, SVH-3, SVH-4, and SVH-5 were grown from such foci. Two cell strains, SVH-1 and SVH-5, were obtained from cell cultures that were selected with G418 for 2 months after transfection. The SVH-2, SVH-3, and SVH-4 cell strains were initiated from cultures of SMCs that were not selected with G418 after transfection. The rate of transformation by either procedure was 1 per 1 06 cells.

Morphology of pSV3-neo-transformed Cells The pSV3-neo-transformed human cell strains consisted of a population of small, refractile, spindle-shaped cells generally arranged in parallel, forming bundles, and a less numerous group of much larger cells (Figure 1). During the proliferative period, mitotic figures were seen frequently, even in areas with multilayered cells. At this stage, the multinucleated cells were rather rare, but as the cell strains approached the 'crisis' stage their frequency increased. The spindle-shaped cells formed circular bundles surrounding a large, multinucleated cell, or an empty space, which suggested that such a cell has detached. In the later part of the 'crisis' stage, most of the cells became extremely large. Some of these cells were multinucleated and showed nuclear budding and micronuclei. Smooth muscle cell cultures from human umbilical cord arteries that were not transfected could be subcultivated for approximately 3 to 5 months, for a maximum of 10 passages. During this period of growth these cells maintained the same spindle-shaped morphology, with regularly oriented bundles. After reaching confluence, the cells formed multilayered hillocks and wavy, transverse ridges of extracellular material59 that were never observed in pSV3-neo-transformed cell cultures. At their last passages, the cells stopped dividing and became very large, but the nuclei maintained a round shape and normal size as the nuclear:cytoplasm ratio decreased

active proliferation, pSV3-neo-transformed cells grown in NCTC 109 medium supplemented with 20% calf serum reached a density of 5 x 1 0 cells/cm2 in 15 days, which was about the double of the number of normal cells grown under similar conditions and approximately 15fold the initial cell number. In medium supplemented with 2% serum, the SVH-1 cell number doubled during the same period. In focus formation assays, SVH-1 cells yielded an average of 30 foci/i 02 cells in medium containing 10% calf serum and 3 foci/102 cells in medium supplemented with 1% serum. The maximum number of passages for normal SMCs grown under similar conditions was 10. Nontransfected human umbilical cord arterial SMCs did not form foci.

Detection of pSV3-neo Sequences Total cellular DNA extracted from the each of five pSV3neo-transformed cell strains was cleaved with Xbal, which has no restriction site in pSV3-neo, and analyzed by Southern blotting. Each of these cell lines produced a smear when probed with radiolabeled pSV3-neo, indicating that the transformed cells retained sequences of different size from the plasmid vector. Short time exposure of the autoradiograms could not demonstrate distinct bands suggestive of monomers of the pSV3-neo. Further analysis demonstrated that these molecules are part of a pool of free recombinant plasmids containing a complete SV40 early region, deleted pBR322 sequences and an insert of cellular DNA (Legrand et al., manuscript submitted for publication). The SVH-1 cell strain was subcloned at passage 4. Total cellular DNA extracted from five of these subclones was digested with Xbal, analyzed by Southern blotting, 1

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Growth of Transformed Cells Five pSV3-neo-transformed cell strains went through a phase of active proliferation during which the cell cultures could be split approximately every 10 days. This period of active growth lasted between 4 (SVH-4 strain) and 9 months (SVH-1 strain) after transfection. The maximum number of passages was 23 for the SVH-1 cell strain (average, 1.56 weeks/passage) and the minimum number was 12 for the SVH-4 cell strain. During this phase of

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artery SMCs (lane 1), human normal tractedfirom umbilical cord skin and 1 SVH- celLs (lane 3) was probed with fibroblasts (lane 2), 32P-labeled 1.1 kb PstIragment ofplasmid 91. The Nordtern blot was exposed to x-ray film for 6 hours.

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and probed with pSV3-neo. Southern blots of subclones 1 and 5 showed a smear identical to that found in the SVH-1 parental cell strain (Figure 2). In subclones 2 and 4, a band of 3.0 kb was detected, and a small amount of pSV3-neo DNA was found in subclone 3. The bands seen in subclones 2 and 4 were of a smaller size than that of pSV3-neo and may represent free plasmids or integrated small fragments of transfected DNA. Digestion with Xbal of total cellular DNA from the SVH-2 cell strain yielded a band of 10.0 kb, which is larger that the transfected plasmid vector (Figure 2B, lane 1, arrow), suggesting the integration of pSV3-neo sequences in the chromosomal DNA. A smear of smaller DNA molecules also was detected. Digestion of SVH-2 cellular DNA with BamHl (Figure 2B, lane 2), which cleaves pSV3-neo DNA in two sites, produced a strong band of 3.0 kb, which represents the SV40 early region from pSV3-neo, and two additional bands of 5.0 and 1.9 kb. These two bands could represent flanking sequences of an integrated SV40 early region sequence. Because the intensity of the 3.0-kb band is higher than that of the Xbal fragment of 10 kb, it appears that several copies of the SV40 early region are integrated in tandem in the SVH-2 cell genome.

Expression of the SV40 Early Region and the p53 Protein Total cellular RNA was extracted from SVH-1 cells and was analyzed by Northern blotting using as probes SV40 DNA or the SV40 early region from pSV3-neo. An intense band of 2.3 kb and two faint bands of 2.1 kb and 2.5 kb, representing the normal-sized transcripts for the SV40 large T and small t antigens, were visible (Figure 3). The presence of the 2.3-kb band indicates an altered transcript for the viral DNA that was confirmed by immunoprecipitation with polyclonal anti-T serum (data not shown). Indirect immunofluorescence and peroxidase staining with monoclonal and polyclonal antibodies showed that almost each pSV3-neo-transformed cell was stained for the large T antigen. As the cells reached the precrisis stage, numerous micronuclei and polylobulated giant nuclei appeared and were stained for SV40 T antigen (Figure 4). Immunostaining of pSV3-neo-transformed cells with monoclonal antibody PAbl 22 showed the presence of moderately strong immunofluorescence with nuclear localization characteristic for the p53 protein (Figure 5).

Chromosome Analysis The chromosome counts in pSV3-neo-transformed cell strains were in the triploid range. A high frequency of structural chromosome aberrations was found in each cell strain examined. The most common chromosome abnormality was dicentric chromosomes. At advanced precrisis passages, dicentric chromosomes were seen in each metaphase of the SVH-1 and SVH-5 cell strains, and many metaphases contained three or four such abnormal chromosomes. Figure 7. Double immunostaining for a-ac-

tin and SV40 T antigen. Every nucleus from the SVH-1 cell culture was stained for the SV40 T antigen but only some of these cells sbowed staining of the actinfilaments, X360.

Expression of the Actin Genes Indirect immunofluorescent staining for smooth muscle a-actin disclosed the presence of bundles of filaments with a regular parallel distribution throughout the cytoplasm in almost every cell from cultures originating from human umbilical cord arteries. Actin filaments were seen only in approximately 20% of pSV3-neo-transformed cells, although all contained the SV40 large T antigen

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Table 1. Content of Cholesterol and Cholesteryl Esters in pSW3-neo Transforrmed Humian Arterial Smooth Muscle Cells Cholesterol Cells Treatment Cholesterol esters Human artery 2± 1 LPDS 48 ± 11 13-VLDL SMCs 45 ± 2 52 ± 5 4±2 LPDS 29 ± 4 pSV3-neo Transformed SMCs 13-VLDL 46 + 3 161 ± 30 The content (g/rmg cell protein) of cholesterol and cholesteryl esters in pSV3-neo transformed human arterial smooth muscle cells (SVH-1 cell strain) incubated for 48 hours with hypercholesterolemic rabbit p-VLDL (50 ,ug protein/ml) or maintained in medium supplemented with lipoprotein deficient serum (LPDS). Data represent mean ± SEM (N = 3).

(Figure 6). Most of the cells that did not display actin filaments were small and spindle shaped. Northern blot analysis of total cellular RNA extracted from SVH-1 cells probed with the Pstl-Pstl fragment of p91 showed only transcripts for the 1- and y-actins, whereas transcripts for the a-actin were present in RNA from Rb-1 cells6O and nontransfected human umbilical cord arterial SMCs (Figure 6).

Lipid Accumulation To examine the capability of the pSV3-neo-transformed cells to accumulate cholesterol, cells were incubated with hypercholesterolemic rabbit 1-VLDL (50 jig protein/ml) for 48 hours and stained with Oil red 0 or Nile red. Both control and pSV3-neo-transformed human arterial SMCs incubated in the presence of LPDS displayed only a few cytoplasmic lipid droplets. In contrast, a massive accumulation of lipid droplets was observed in pSV3-neo-

transformed human SMCs. The lipid droplets were present in almost all the cells, and most of them were localized in the perinuclear region. Chemical analysis showed trace amounts of cholesteryl esters in normal arterial SMCs or pSV3-neotransformed cells maintained in medium supplemented with LPDS, whereas pSV3-neo-transformed cells exposed to medium supplemented with 1-VLDL accumulated significantly higher amounts of cholesteryl esters than found when normal human SMCs were incubated in similar medium (Table 1). Interactive laser cytometry showed a significant increase in the amount of intracellular Nile red-stained material after treatment of pSV3-neo-transformed SMCs with hypercholesterolemic rabbit 1-VLDL. In a typical experiment, the average fluorescence for SVH-1 cells incubated in the presence of LPDS was 380.4 ± 92.4. The average fluorescence of these cells incubated in medium supplemented with f-VLDL was 1085.5 ± 306.1 (Figure 8). Flow cytofluorometry of SVH-1 cells incubated with 1-VLDL showed a similar significant increase in fluorescence (Figure 9). In both analyses, the histograms indicate a normal distribution for the lipid accumulation in these cells. The increase in intracellular lipid loading after incubation with atherogenic lipoproteins was consistently found throughout the life span of transformed cells.

Discussion We report here that the transfection of the early genetic region of SV40 into human arterial SMCs resulted in the formation of colonies of morphologically transformed Figure 8. Computer display of quantitative lipidfluorescence analysis. The cross-batched histogram represents a population of SVH-1 cells incubated with medium supplemented uwith LPDS and scanned with ACAS 570. The filled-bars histogram represents a population of cells incubated uwitb rabbit 1-VLDL (25 pg protein/ml).

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cells. These foci were amplified into cell strains that exhibit properties typical for SV40-transformed cells,39 such as changes in morphology, growth, and karyotype. The transformed cells contain virus DNA sequences and express viral transcripts and virus-specific antigens. After several months of increased proliferation, the cells eventually reach a stage of 'crisis,' and terminate their life span.

Like most normal human cells, SMCs cultured from uninvolved arteries have a finite in vitro life span61 '- that is significantly prolonged by SV40-induced transformation. A role for the SV40 T antigen in the initiation and maintenance of transformation has been demonstrated clearly in different types of cells.63 Among multiple other actions,64 the T antigen appears to inactivate specific proteins that control proliferation,65 such as the retinoblastoma susceptibility gene (Rb) product66 and the p53 phosphoprotein.67 The p53 protein is detected in pSV3neo-transformed arterial human SMCs, but the significance of its presence remains to be elucidated. Further studies could establish whether vascular SMCs would overexpress this protein in response to other mitogenic stimuli. As has been the case for most SV40-induced transformation of human cells,`23963 the expression of the large T antigen and the accumulation of the p53 protein in pSV3-neo-transformed vascular SMCs did not result in immortalization. These results support previous data indicating that SV40 T antigen alone is not sufficient to maintain immortalization in human cells.32'6 The extended in vitro life span of pSV3-neo-transformed human SMCs suggests that, like in human fibroblasts, the SV40 T antigen activity was incompetent to affect mechanisms

that control crisis and cell death, but was able to bypass a hypothetical mechanism that controls cellular senescence.63-9 The features displayed by the pSV3-neotransformed human arterial SMCs may provide an interesting model for the study of the mechanisms in control of vascular SMCs proliferation and senescence. A reduction in a-actin transcripts and filaments was found in pSV3-neo-transformed human SMCs. Actincontaining material was reduced after SV40 transformation of 3T3 and rat cells,70 and this was related to the expression of the SV40 early genetic region,71 particularly the small t antigen.72 The role of the small t antigen remains controversial, however.73 Cultured vascular SMCs undergo differential expression of isoactins in relation to their growth state.74 A decreased actin content and a switch in the pattern of actin isoforms expression occur in the proliferative early stage of experimental intimal thickening and in human atheromatous plaques.75'76 It appears that evaluation of cytoskeletal changes in SMCs under various conditions could provide useful information as to the alterations of gene expression during experimental and human atherogenesis. A remarkable feature of pSV3-neo-transformed human vascular SMCs is the accumulation of large amounts of cholesteryl esters after exposure to atherogenic Iipoproteins. Accumulation of different amounts of cholesteryl esters was observed in clones of pSV3-neotransformed rabbit aorta SMCs incubated with atherogenic rabbit 1-VLDL,51 but some SV40-transformed clones showed lipid accumulation even in the absence of lipoproteins.77 Simian-virus-40transformed human keratinocytes show similarities in the expression of LDL receptors and the regulation of cholesterol metabolism with

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uninfected cells,78 but in SV40 transformed rat fibroblasts, overexpression and decreased down-regulation of LDL receptors are found.79 Significantly greater amounts of saturated cholesteryl esters and triacylglycerols accumulate in human arterial SMCs infected with HSV type 1, and this effect was due in part to decreased cholesterol ester hydrolysis.80 Based on these studies, subclonal populations of pSV3neo-transformed human SMCs may possess several mechanisms that are responsible for the lipid overloading observed in the presence of atherogenic lipoproteins.

Acknowledgments The monoclonal antibody PAb901 was a gift from Dr. Satvir S. Tevethia, Department of Microbiology, Hershey College of Medicine, Pennsylvania State University, Hershey, Pennsylvania. The rabbit polyclonal antibody against SV40 T antigen was provided by Dr. Janet Butel, Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas. The authors thank Dr. Carol Pillinger and the Department of Obstetrics and Gynecology, Lexington Medical Center, South Carolina, for their assistance in obtaining human umbilical cords. They also thank Dr. Richard Hunt for allowing the use of the ACAS 570 laser cytometer and Ms. Indhira Handy for her assistance in carrying out the digital imaging fluorescence microscopy and the flow cytometry.

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