The synthesis of surface coat materials in normal and transformed cells

The rate of synthesis and thickness of the surface coat material in a range of virus-transformed and chemically-transformed cell lines were measured by ellipsometry. Cell lines transformed by polyoma virus, SV 40 virus, Rous sarcoma virus and murine sarcoma virus had a significantly thicker coat than the normal parent cells. An increase in the thickness of the cell coat was not a consistent feature of the transformed cell state since this change was not detected in cell lines transformed by methylcholanthrene. The rate of synthesis of the surface coat was significantly faster in transformed cells than in normal cells. Coat synthesis in normal and transformed cells was inhibited rapidly by treatment with cycloheximide. Inhibition of cellular RNA synthesis by actinomycin D produced rapid inhibition of coat synthesis in normal and chemically-transformed cell, but in certain virus-transformed cell lines coat synthesis continued for up to h. The significance of these changes in the pattern of coat synthesis in transformed cells in relation to their altered surface properties is discussed.     

Agglutination of Normal Cells by Plant Lectins following Infection with Nononcogenic Viruses

MAMMALIAN cells transformed by oncogenic viruses and chemical carcinogens undergo characteristic changes in their surface properties, some of which affect the control of cell multiplication. Certain plant lectins agglutinate transformed cells but not normal cells^1–6, which, although possessing binding sites, can only be agglutinated following treatment with proteolytic enzymes^3–5. Furthermore, both normal and transformed cells bind equal amounts of lectins, indicating that the increased susceptibility of transformed and trypsinized cells to agglutination is not caused by simple “unmasking” of hidden receptor sites. Nevertheless, the increased susceptibility of normal cells to agglutination following trypsinization may well result from changes occurring in the cell coat material. Since lytic infections with certain nononcogenic viruses¹⁰ and various drug treatments¹¹ are known to cause modification of the coat material in normal cells, we were interested to see whether these treatments increased the susceptibility of cells to agglutination by lectins.     

Differences in sialic acid contents of low cancer cells,high cancer cells and normal mouse lung counterparts

Sialic acid contents of low cancer (P 4 BIS) high cancer (P 4 BIS T) cells and their normal (PB) mouse lung counterparts have been determined. This content is 5 to 10 fold higher for cells in logarithmic phase growth than for confluent cells, as well for normal cells as for transformed derived cells lines. Growing normal PB cells contain a large amount of sialic acid (21.2 μg/10⁶ cells): it is reported that cellular sialic acid content decreases dramatically with the tumor producing capacities of the cells (3.4 μg/10⁶ P 4 BIS cells; 2.1 μg/10⁶ P 4 BIS T cells).It has been found in conditions which maintain cell viability that transformed neuraminidase treated cells or trypsin treated cells liberate large percentages of sialic acid, or sialoglycoproteins, whereas small percentages are liberated from normal cells, indicating that transformed cell surface glycoproteins may be reached more easy by enzymes that normal cells: in that aspect low cancer cells (P 4 BIS) appear transitory between normal (PB) and high cancer cells (P 4 BIS T) in the same way they are transitory in tumor producing capacities.     

Binding of Concanavalin A to Normal and Transformed Cells as Detected by Immunofluorescence

CHANGES in the nature and conformation of the cell membrane have been considered to play a significant role in malignant transformation¹. Transformed cells have been reported to have altered surface properties detectable by the binding of some plant derived lectins leading to agglutination^2–5. It has been suggested that the surface of transformed cells contains exposed sites where these agglutinins interact, whereas normal cells have such sites in a cryptic form^6,7. I now report evidence for the binding of the phytagglutinin concanavalin A (Con A) on the surface of both normal and transformed cells as detectable by immunofluorescence.     

Agglutination of Normal and Rous Sarcoma Virus-transformed Chick Embryo Cells by Concanavalin A and Wheat Germ Agglutinin

RODENT cells in culture transformed by oncogenic DNA viruses have surface sites that on normal cells are usually present in latent form only^1,2. This difference in surface properties can be detected by plant glycoproteins such as wheat germ agglutinin (WGA) and concanavalin A (Con A), which agglutinate only transformed cells, because they have certain carbohydrate moieties on their neoplastic surfaces^1–4. According to some investigators, normal and neoplastic cells that have been freshly isolated also exhibit this marked difference^3,5; according to others^6,7, there is no such distinction. We have looked for such differences in cells transformed by RNA tumour viruses and in several types of normal and naturally occurring malignant cells and their normal counterparts.     

External cell surface protein phosphorylation in normal and Rous sarcoma virus transformed chick embryo fibroblasts

Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (γ³²P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, ³²P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by tryspin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on ³²P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphoryllated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein physphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.     

Effect of Interferon on Some Aspects of Transformation by Polyoma Virus

WHEN BHK 21 hamster cells are infected with polyoma virus¹, there is no vegetative growth of virus, but stably transformed cells appear. These transformed cells are more easily transplanted than BHK 21 cells; they initiate their growth cycle in otherwise restrictive cultural conditions such as the absence of serum, high density and suspension; they grow with random orientation and have exposed on their surfaces receptor sites for certain glycoprotein agglutinins^2–5. The proportion of stably transformed cells is low, even after high doses of virus. But a much higher proportion (sometimes all cells) shows abortive transformation—changes characteristic of transformation, but which last only a few days. In suspension cultures, for example, most of the infected cells grow into small colonies of four to thirty-two cells⁶. In surface cultures deprived of serum DNA synthesis is initiated and the cells may then divide at least once⁷: they also temporarily lose the normal parallel orientation and develop the typical random appearance of transformed cells. Moreover, the polyoma nuclear T-antigen and also a surface antigen detected by immunofluorescence, appear temporarily in most polyoma infected BHK 21 cells⁸, while 3T3 cells exposed to SV40 virus show transient exposure of cell surface sites reacting with conconavalin A (ref. 9).     

The cell coat of the olfactory epithelium proper and vomeronasal neuroepithelium of the rat as revealed by means of the Ruthenium-red reaction

Summary The apical cell coat of the olfactory epithelium proper and the vomeronasal neuroepithelium of the rat was investigated electronmicroscopically by means of the Ruthenium-red reaction. In the olfactory epithelium proper, the cilia of receptor cells and microvilli of supporting cells possess a cell coat measuring approximately 10 nm in thickness. In the vomeronasal neuroepithelium, the apical cell coat is thicker than in the olfactory epithelium proper. On microvilli of vomeronasal receptor cells the cell coat varies in thickness from 15 to 20 nm, and on microvilli of supporting cells it measures approximately 75 nm. The functional implications of these findings are discussed.A portion of this study was presented at the 6^th European Anatomical Congress in Hamburg. This publication is dedicated to Prof. E. KlikaSupported by the Deutsche Forschungsgemeinschaft (Br 358/5-1).     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Correlation of (Na---K)-ATPase activity with growth of normal and transformed cells

The (Na⁺---K⁺)-stimulated Mg²⁺-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na⁺---K⁺)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na⁺---K⁺)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.     

Comparison of the mechanical properties of normal and transformed fibroblasts

In order to achieve coordinated migration through extracellular matrix and endothelial barriers during metastasis, cancer cells must be endowed with specific structural and adhesive properties. In this context, comparison of the mechanical properties of transformed versus normal cells, on which little quantitative information is available, was the focus of this study. Normal human dermal fibroblasts and their SV40-transformed counterparts were analyzed using various manipulations. First, micropipet aspiration of suspended cells allowed calculation of a cortical tension (similar for normal and transformed cells), and an apparent viscosity (30% lower for transformed than for normal fibroblasts); in addition, transformed fibroblasts exhibited a more fragile surface than their normal counterparts. Second, tangential ultracentrifugation of adherent cells demonstrated cellular elongation in the direction of the centrifugal field and the existence of critical forces for cell detachment, around 10⁻⁷ N: these were 1.6-fold greater for normal than for transformed cells. Finally, examination of the wrinkle patterns formed by cells plated on a deformable polydimethylsiloxane substrate, plus analysis of cell retraction caused by ATP treatment following detergent permeabilization showed that normal fibroblasts exhibited much more contractility than their transformed counterparts, which we characterized by a cell contraction rate. Such quantitative parameters which reveal differences in the mechanical behavior of normal and transformed cells may be used in the future as new markers; of oncogenic transformation.     

Calcium effects on epidermal growth factor receptor-mediated endocytosis in normal and SV40-transformed human fibroblasts

Lowering of extracellular Ca²⁺ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus₄₀(SV₄₀)-transformed WI38 cells do not exhibit this Ca²⁺-dependent arrest. One possibility for this difference in Ca²⁺ requirement is that extracellular or surface membrane-bound Ca²⁺ may be required for growth factor receptor-mediated endocytosis and this Ca²⁺ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca²⁺ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV₄₀ WI38 cell. The binding of [¹²⁵I]EGF to the cell surface is not significantly altered by lowering of Ca²⁺ to 10^?5-M levels in either the normal or transformed cell. At this Ca²⁺ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca²⁺ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca²⁺. A twofold reduction in the rate of release of acid-soluble ¹²⁵I occurs in the normal but not the transformed cell under conditions of lowered medium Ca²⁺. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca²⁺-dependent regulator protein calmodulin, causes an inhibition of [¹²⁵I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [¹²⁵I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.     

Relationship between cell surface protease activity and doubling time in various normal and transformed cells

A sensitive method for measuring cell surface and secreted protease activity utilizing ³H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloroacetic acid soluble ³H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3–4 times more proteolytic activity than the normal cells.Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.     

The glycoprotein surface coat on different classes of murine lymphocytes

Both thymus and spleen lymphocytes of mice have been shown to possess a surface coat visible when the cells are stained with ruthenium red. Measurements on high-magnification photographs showed that the coat on thymus lymphocytes is significantly thicker than on spleen lymphocytes from genetically athymic nu/nu mice, which form a pure B cell population. Most of the coat on thymus cells is removed by treatment of the cells with neuraminidase or with trypsin, indicating that the coat is glycoprotein in nature. Functional implications of the difference in the cell coats of thymus and B cells are discussed.     

Effect of low extracellular Ca2+ on growth spreading area,cytoplasmic Ca2+ concentration,and intracellular pH in normal and transformed human fibroblasts

The transformation of certain cells reduces the requirement of extracellular Ca²⁺ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca²⁺ than normal WI-38 cells. Spreading area of normal cells decreases when cultured in 10 μM Ca²⁺ medium. Intracellular calcium concentration ([Ca²⁺]_i), of the normal and transformed cells cultured in 10μM and 2 mM Ca²⁺ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca²⁺], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca²⁺],_i of both normal and transformed cells. Although the total decrease in [Ca²⁺]_i is the same for both cell, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca²⁺ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca²⁺]_i value in both cells. In addition, we investigated, using BCECF as a fluorecent probe, the intracellular pH (pH_i) of normal and transformed cells maintained at low and normal Ca²⁺. The low Ca²⁺ condition makes pH_i acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attacment, followed by the reduced spreading area, induced the acidic pH_i. These results suggest that the reduced Ca²⁺ requirement of transformed cells for growth is related to the mechanism of pH_i regulation rather than Ca²⁺ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells. © 1993 Wiley-Liss, Inc.     

A consideration of the role of cell surface macromolecules in the process of viral transformation

Summary There is extensive physiological evidence implicating the cell surface as the key organelle which mediates the cell:cell interactions which underlie both normal and neoplastic growth. This information has now been supplemented with biochemical and biophysical data which indicates that surface macromolecules, in particular the heteroglycans of transformed cells, differ from those which lie at the periphery of normal cells. In the case of cells neoplastically transformed by most tumour viruses it is clear that the small virus genome (2–5×10⁶ daltons) cannot carry the total genetic information to accomodate these various biochemical modifications, if indeed they are encoded in separate genes (1). To examine the part played in transformation by cellular genes coding for surface heteroglycan formation, we have turned to a study of SV-3T3 cells (ts H6-15) which are temperature-sensitive for expression of the transformed cell phenotype (2). The data show that cells grown under conditions permissive and non-permissive for such expression exhibit the same pattern of formation of glycolipids, and of the majority of the polypeptides of the plasma membrane. There are, however, significant differences in the synthesis of some glycopeptides. A large molecular weight, trypsin-labile glycopeptide, present at the surface of untransformed fibroblasts but barely measurable in some of their virus-transformed derivatives (3), was detected, essentially at the same level, at the surface ofts H6-15 cells grown at the permissive and non-permissive temperatures. The significance of these observations is discussed. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

1H and 13C nuclear magnetic resonance spectra of the lipids in normal and SV 40 virus-transformed hamster embryo fibroblast membranes

Well resolved ¹H and ¹³C NMR spectra were obtained with normal and SV 40-transformed cell membranes. Estimation of the ratio of ¹³CT₂ values of the normal to transformed cell membranes showed an increased intermolecular motion in the transformed cell membranes. The temperature dependence of the (CH₂)_n line in the ¹H spectra in the temperature range 298–343 °K shows an activation energy for the lateral diffusion of the fluid phospholipid regions in the normal cell membranes while the transformed ones show practically no temperature dependence in this temperature range. The fluidity of the phospholipid region in the transformed cell membrane seems to be significantly higher than that observed in the normal cell material. These data support and extend the findings concerning the mobility of the concanavalin A binding/agglutinating sites on the surface of normal and virus-transformed cells and suggest further approaches to the study of the membrane alterations in tumor cells.     

Electron microscopy of cell surfaces of Paramecium caudatum stained with ruthenium red

The cell surface of Paramecium caudatum, syngen 3, was examined by electron microscopy using ruthenium red (RR) staining. The RR-positive surface coat is of uniform thickness and is found on the entire surface of paramecia, including the gullet area. The surface coat is also observed on the membranes in the tightly united region of conjugating cells. Measurements of the thickness of the surface coat in six stocks of P. caudatum demonstrate a significant difference between complementary mating types: cells of mating type VI have a thicker layer than those of mating type V. No detectable differences in morphology of the surface coat are observed between mating reactive and unreactive cells. Observation in detached cilia indicated that changes in the morphology of the surface coat provoked by the detachment procedure have no effect on mating reactivity. RR stainable substances are detected on both sides of ciliary membranes.     

Effect of proteolytic inhibitors on growth and surface architecture of normal and transformed cells

Protease inhibitors were tested for their effect on the growth of normal and SV40-transformed mouse fibroblasts. The protease inhibitors TAME¹ and EWTI¹, which act competitively on proteases, reduce the growth of transformed cells more than that of untransformed parent cells. However, transformed cells grown in medium containing these drugs do not show contact inhibition of cell division or decreased agglutinability with Concanavalin A. The inhibition of growth is due to an extended duration of all phases of the cell cycle. The protease inhibitor TLCK¹, an active site titrant reacting irreversibly with trypsin, blocks transformed cells in the premitotic stage of the cell cycle. This effect does not occur in the untransformed parent cells. The decrease in agglutinability of transformed cells treated with TLCK is correlated with a partial synchronisation in the G2 stage of the cell cycle. Our results do not support the hypothesis that protease inhibitors induce transformed cells to assume a normal growth pattern and that this is accompanied by a decreased agglutinability with plant lectins.     

Non-lytic, non-ionic detergent extractions of plasma membrane constituents from normal and transformed fibroblasts

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cells employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, ⁵¹Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodina- tion followed by detergent extraction permits demonstration of a 100 000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.