A Scanning Electron Microscope Study of Surface Features of Viral and Spontaneous Transformants of Mouse Balb/3t3 Cells

In culture medium containing heparinized, heatinactivated, chicken plasma, normal chicken heart mesenchymal cells do not proliferate but their Rous sarcoma virus-infected counterparts proliferate maximally. In medium containing serum derived from chicken whole blood or plasma, on the other hand, normal chicken heart mesenchymal cells proliferate actively, at similar overall rates and to similar extents. The rate and extent of normal cell proliferation are decreased by a factor of approximately 'A with whole blood-derived serum that is heparinized and inactivated; proliferation ceases in plasma-derived serum that is heparinized and inactivated. Heparinization and inactivation of serum does not affect the proliferation of Rous sarcoma virus-infected cells, indicating that this combined treatment eliminates a mitogenic (regulatory) rather than a supportive (nutrient) factor(s) for cell replication. We hypothesize that mitogen(s) is released from plasma protein precursors when plasma clots in the presence of formed elements of the blood or when plasma-derived serum is exposed to cultured cells; heparinization and inactivation, within the framework of this hypothesis, would render nonfunctional the plasma protein precursor(s) from which the mitogen(s) is generated. Alternatively, our data are consistent with the release of two mitogens during blood clotting, one from plasma protein precursors and the other from formed elements of the blood. We also have studied the proliferative behavior of Swiss and BALB/c 3T3 cells in whole blood-derived and plasma-derived human serum. Our studies suggest that the platelet-derived growth factor has an artifactual supportive (nutrient) role, rather than an authentic mitogenic role, in cell replication.Between 1971 and 1973, Balk and coworkers (1-3) demonstrated that normal chicken pectoral muscle fibroblasts did not proliferate in low-calcium culture medium containing chicken plasma but proliferated actively when this medium contained chicken serum. On the basis of that observation, it was postulated that a mitogen might be released from plasma protein precursors or from formed elements of the blood during the clotting process and that this mitogen, found in serum but not in plasma, might represent a "wound hormone" capable of initiating cell proliferation at sites of tissue injury. Because of uncertainties inherent in the interpretation of data derived from studies using a culture medium with a low calcium concentration (4), however, further studies of the serum-plasma difference were deferred until a culture system was available in which normal cells were quiescent in a plasma-containing medium with physiological ion concentrations.Other workers subsequently have claimed that the mitogenic property present in serum, but not plasma, resided in a specific polypeptide "platelet-derived growth factor" (for general re-views, see refs. 5 and 6; for critical reviews, see refs. 7 and 8). Ross and Vogel (5) Because the activity ofthe platelet-derived growth factor has been defin...

Section: Resultssupporting

confidence: 79%

Mitogenic factors present in serum but not in plasma.

Balk¹,

Levine²,

Young³

et al. 1981

“…The fibrinolytic activity exhibited by the established endothelial cell line seems to reflect a physiological characteristic of the intimal layer from which it is derived rather than a property acquired during the culture period as a result of spontaneous transformation because: (i) the growth pattern and the morphology of the established endothelial cell line are the same as those of the primary culture from which it is derived; in contrast, spontaneous transformants of Balb/3T3 cell line [an embryonic mouse cell line of endothelial derivation (21)] are distinctly different from the nontransformed parental line with respect to cell surface and growth pattern (21); and (ii) the endothelial cell line from rabbit aorta is not tumorigenic when tested in the immunologically incompetent (athymic) mouse. The fibroblast line (a normal, nontransformed cell line as demonstrated by its inability to form tumor in the nude mouse) has no significant fibrinolytic activity.…”

Section: Discussionmentioning

confidence: 99%

Hormone and neurotransmitter receptors in an established vascular endothelial cell line.

Buonassisi¹,

Venter²

1976

229

A cell line from the intima of the rabbit aorta has been established. This cell line exhibits strict contact inhibition, and morphologically resembles intimal endothelial cells. B-type blood group antigens and the presence of fibrinolytic activity also distinguish these cells from smooth muscle cells and from fibroblasts of the aortic wall. Endothelial cells were assayed for changes in levels of adenosine 3': 5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) in response to a series of vasoactive drugs. Control levels for cAMP and cGMP are 7.01 4 0.82 and 1.50 4 0.06, respectively (mean 4 SEM). Norepinephrine, acetylcholine, 5-hydroxytryptamine, and phenylephrine increased the levels of both nucleotides significantly. Propranolol (10-5 M) and phentolamine (10-5 M) inhibited, respectively, the cAMP and cGMP response to norepinephrine. Angiotensin II and histamine significantly increased cGMP levels but not cAMP levels of the endothelial cells. The cGMP increases with acetylcholine were inhibited by atropine. These results indicate that the established cell line is endothelial in nature and contains cellular receptors to a variety of vasoactive agents. The role traditionally assigned to the endothelial cell has been that of a rather passive lining of the lumen of the vessels. Recent The line of rabbit fibroblasts used for these studies has been described (6).Mixed Agglutination Test for Blood Group Antigens. Mixed agglutination tests on cells growing in plastic petri dishes were done according to the technique described by Franks and Dawson (7). Antisera were purchased from the Dade Division of the American Hospital Supply Co.Scanning and Transmission Electron Microscopy. To prepare the cultures for scanning electron microscopy, we grew the cells on cover slips (12 mm diameter). At confluence, the cells were first fixed at 370 for 1 hr with 1% glutaraldehyde buffered with 0.1 M cacodylate at pH 7.2, followed by fixing with 3% glutaraldehyde buffered with 0.1 M cacodylate at pH 7.2 for 1 hr at room temperature. After rinsing the cells with distilled water, they were dehydrated in an alcohol series (30-100%) and critical point dried (8). The cells were coated with a thin layer of palladium gold (200 A) in a vacuum evaporator and then viewed under a Cambridge S4 scanning electron microscope.The technique of preparing the cells for transmission by means of electron microscopy has been reported (6). Cultured endothelial cells were also examined by transmission electron microscopy after the second passage.

“…Fig. 4 is actually a picture of a cell incubated with 5 ,gg/ml of cycloheximide from the time of the shift from 410 to 36' until fixation 2 hr later. In a control experiment we have shown that under these conditions the level of incorporation of radioactive leucine into protein in the presence of cycloheximide is only 1% of that occurring in its absence.…”

Section: Resultsmentioning

confidence: 99%

Surface ruffles as markers for studies of cell transformation by Rous sarcoma virus.

Ambros¹,

Chen²,

Buchanan³

1975

Confluent chick embryo fibroblasts infected with the Ts68 mutant of Rous sarcoma virus were examined by scanning electron microscopy at the permissive (360) and nonpermissive (410) temperatures for transformation. Infected cells shifted from 410 to 360 undergo a change in shape from elongated to rounded. This process is preceded by the appearance of surface ruffles on the cell. These surface ruffles are not observed on cells maintained at 410, appear as early as 0.5 hr after a shift to 360, and are common on cells maintained at 360. By 3.5 hr after the shift from 410 to 360, cultures appear fully transformed by the criteria of cell roundedness and the presence of surface ruffles. This surface alteration of cells is the earliest event of those so far reported during the transformation process and is not dependent upon protein synthesis and extracellular plasminogen during the period of temperature shift. Transformation of cultured cells by RNA tumor viruses is accompanied by changes in morphology and other biochemical functions. When chick embryo fibroblasts are infected with Kawai and Hanafusa's Ts68 mutant of the SchmidtRuppin strain (subgroup A) of Rous sarcoma virus (RSV), the expression of the transformation phenotype of infected cells is dependent upon temperature, i.e., permissive at 360 and nonpermissive at 410, whereas other viral related functions are reported to be the same (1). Alterations in several transformation-related biochemical functions in Ts68-infected cells have been reported during the time course of a temperature shift from 410 to 360. In brief, there was an increase in glucose uptake from 4 to 6 hr; loss of membrane-associated protein of molecular weight 45,000 from 3 to 6 hr (2); secretion of proteases from 8 to 20 hr (3); and reduction in a surface protein of 250,000 daltons (LETS or Z protein) after 20 hr (4).However, the most remarkable observation has been the rapid change in cell morphology from a flattened and elongated to a more rounded, refractile shape during temperature shift. This significant change can be detected by use of the light microscope as early as 1.5-6 hr (1,4 (5). Cultures were not washed prior to fixation to avoid dislodging poorly attached cells. The medium was sucked off and a glutaraldehyde solution, 2.5% in 0.05 M cacodylate and half-strength phosphate-buffered saline at pH 7.2, was added gently to the culture dish containing the coverslip for 20 min. Cultures were then washed in 0.1 M cacodylate, postfixed for 20 min in 2% osmium tetroxide buffered with 0.1 M cacodylate at pH 7.2, and finally washed with distilled water. The cells were then subjected to sequential dehydration by transfer through a graded series of water/acetone and then acetone/amyl acetate mixtures, and finally dried at the critical point of CO2 in a critical point dryer. Specimens were stored in a desiccator under vacuum until coated with a 200-300 A film of gold. The preparations were viewed in a JSM-U3 scanning electron microscope at 25 kV. Transmission electron micrographs were m...