Transformation-induced alterations in adhesion : Binding of pre-formed cell aggregates to cell layers

The formation of intercellular adhesions by mouse 3T3 cells and their SV40-transformed derivatives is analysed by measuring the binding of pre-formed aggregates of these cells to cell layers or to a plastic substratum. The rationale for this procedure is to reduce the effects of cell dissociation on quantitative assessments of adhesive interactions. The fibroblasts within the aggregates retain the growth characteristics these cells show in monolayer culture. The proportion of aggregates binding is independent of the number of aggregates added and changes with time in a manner consistent with a first-order process, allowing the percent aggregates binding per unit time to serve as a parameter of intercellular adhesion. The rate of binding in homologous adhesive interactions is slower than in heterologous ones, binding in 3T3SV interactions is slower than in 3T3 interactions, and binding to cellular substrata is slower than to plastic. Binding of 3T3SV aggregates is readily distinguished from binding of 3T3 aggregates by the presence of a brief lag in binding rate, the formation of irregular projections from the bound aggregate, and a differential effect on binding rates of varying the temperature or of treating a single reactant with glutaraldehyde. Thus, there are quantitative and qualitative differences in the adhesive interactions of normal and transformed cells. The distinct binding properties of 3T3SV aggregates and the greater binding rates in heterologous interactions may be relevant to the invasive behavior of transformed cells in vivo.     

Cell adhesion and cell surface topography in aggregates of 3T3 and SV40-virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy

A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.     

AN ASSAY FOR INTERCELLULAR ADHESIVE SPECIFICITY

A modification of an assay for intercellular adhesive specificity is described. The method involves the collection of radioactively labeled cells by aggregates of the same (isotypic aggregates) or different (heterotypic aggregates) types of tissue and determination of the number of cells collected by liquid scintillation counting. The use of ³²P to label the tissues permitted a much more rapid estimation of cell collection than was obtained previously. With the use of chick embryo neural retina, liver, forebrain, pectoral muscle, and heart ventricle tissue, it was shown that isotypic was always greater than heterotypic collection. Labeled neural retina cell collection by neural retina aggregates was studied as a function of time, cell suspension density, aggregate diameter, temperature, and aggregate number. Neural retina aggregates were treated with certain enzymes in an attempt to determine whether specific changes on the surface of the aggregates would interfere with labeled neural retina cell collection. Of the various proteases and glycosidases tested, only β-galactosidase rendered the surface more nonspecific.     

Simian virus 40 rapidly lowers cAMP levels in mouse cells

The addition of SV40 to contact inhibited $\text{Balb}\text{3T3}$ cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.     

Specificity of cell-cell interactions in sea urchin embryos: Appearance of new cell-surface determinants at gastrulation

Studies on normal and hybrid sea urchin embryos show that, beginning at gastrulation, hybrid cells express cell-surface antigens specific to both species. The appearance of these antigens is shown to be correlated with a change in the adhesive specificity of hybrid cells: Beginning at gastrulation, hybrid cells recognize and adhere to embryonic cells of both normal genotypes. Prior to gastrulation, hybrid cells adhere to cells of the maternal genotype only. Two adhesion assays demonstrate these adhesive preferences. (i) When cell aggregates are placed together in a dish, Lytechnius aggregates fuse together, and Tripneustes aggregates fuse together, but aggregates of the two species do not fuse with each other. Hybrid cell aggregates, if they are past the beginning of gastrulation, fuse to both Tripneustes and Lytechinus aggregates. (ii) In a collection assay, midgastrula cells of the hybrid embryos are collected at a high rate to aggregates of either species. Pregastrula hybrid cells collect at a high rate to aggregates of the maternal species only. This change in adhesive preference is temporally correlated with the appearance of new cell surface antigens. Antiserum was prepared in rabbits against membranes from Lytechinus gastrulae. Indirect immunofluorescence tests show that hybrid cells of the cross (T♀ × L♂) express Lytechinus-specific antigens at the cell surface beginning at gastrulation. Furthermore, an apparent relationship between the new cell-surface antigens and adhesion exists in that Lytechinus cell adhesion is inhibited specifically after binding Fab fragments of the Lytechinus antiserum. The antiserum has no effect on Tripneustes adhesion. The Lytechinus adhesion-inhibiting activity can be removed by absorption of the antiserum with Lytechinus cells.     

Intercellular adhesion mediated by human muscle neural cell adhesion molecule: effects of alternative exon use

Mouse 3T3 fibroblasts were permanently transfected with cDNAs encoding isoforms of the neural cell adhesion molecule (N-CAM) present in human skeletal muscle and brain. Parental and transfected cells were then used in a range of adhesion assays. In the absence of external shear forces, transfection with cDNAs encoding either transmembrane or glycosylphosphatidylinositol (GPI)-linked N-CAM species significantly increased the intercellular adhesiveness of 3T3 cells in suspension. Transfection of a cDNA encoding a secreted N-CAM isoform was without effect on adhesion. Cells transfected with cDNAs containing or lacking the muscle-specific domain 1 sequence, a four-exon group spliced into the muscle but not the brain GPI-linked N-CAM species, were equally adhesive in the assays used. We also demonstrate that N-CAM-mediated intercellular adhesiveness is inhibited by 0.2 mg/ml heparin; but, at higher concentrations, reduced adhesion of parental cells was also seen. Coaggregation of fluorescently labeled and unlabeled cell populations was performed and measured by comparing their distribution within aggregates with distributions that assume nonspecific (random) aggregation. These studies demonstrate that random aggregation occurs between transfected cells expressing the transmembrane and GPI-linked N- CAM species and between parental cells and those expressing the secreted N-CAM isoform. Other combinations of these populations tested exhibited partial adhesive specificity, indicating homophilic binding between surface-bound N-CAM. Thus, the approach exploited here allows for a full analysis of the requirements, characteristics, and specificities of the adhesive behavior of individual N-CAM isoforms.     

Dibutyryl cyclic AMP treatment of 3T3 and SV40 virus-transformed 3T3 cells in aggregates. Effects on mobility and cell contact ultrastructure

The random cell movement of BALB/c 3T3 and SV40 virus-transformed BALB/c 3T3 cells within homogeneous aggregates was studied by observing the degree of penetration of newly attached [3H]thymidine-labeled cells into the interior of the aggregates. The 3T3 cells penetrated into 3T3 aggregates an average of 0.89 cell diameter in 1.5 days, whereas the SV40-3T3 cells penetrated into SV40-3T3 aggregates an average of 3.20 cell diameters in the same time. Treatment of the aggregates with theophylline, theophylline plus prostaglandin E1, or theophylline plus dibutyryl cyclic AMP all decreased the penetration of the SV40-3T3 cells into SV40-3T3 aggregates (2.36, 1.22, and 0.79 cell diameters, respectively). The same treatments had little effect on 3T3 aggregates. The ultrastructure of 3T3 and SV40-3T3 cells in aggregates was examined by transmission electron microscopy. The 3T3 cells in aggregates were surrounded by microvilli and lamellipodia which were in contact with neighboring cells, whereas SV40-3T3 cells were nearly devoid of microvilli and lamellipodia and made contact at broader, less regular surface undulations. Treatment with theophylline plus dibutyryl cyclic AMP resulted in the appearance of microvilli on SV40-3T3 cells and also appeared to increase the area of intercellular contacts in both 3T3 and SV40-3T3 cells. These observations were supported for the surface cells of the aggregates by scanning electron microscopy.     

Specificity of cell adhesion: differences between normal and hybrid sea urchin cells.

The adhesive specificity of embryonic sea urchin cells from two species, and the two hybrid crosses between these species was examined by a cell-aggregate collection assay. Cells of normal Lytechinus or Tripneustes embryos were found to adhere to homospecific cell aggregates at a much higher rate than they would adhere to heterospecific aggregates. Hybrid cells adhered to collecting aggregates at an intermediate rate. The observed pattern of hybrid cell adhesion suggested that paternal gene products are capable of modifying cell surface adhesive sites as early as the mesenchyme blastula stage.     

Inactivation of SV40 replication by derivatives of benzo[a]pyrene.

When African green monkey kidney cell lines, infected with simian virus 40, were exposed to benzo[a]pyrene-7,8-dihydrodiol or $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, inhibition of progeny virus formation was observed. Alkylation of SV40 DNA with $\text{anti}$-BPDE inhibits the infectivity of this viral DNA; however, the inactivation does not follow a single-hit mechanism. Studies on [³H]thymidine incorporation indicate that SV40 DNA synthesis is markedly impaired for the first 12 hours following BPDE treatment; 24 to 36 hours later, however, SV40 DNA synthesis is almost normal. These data suggest that the inhibition of SV40 DNA synthesis by BP derivatives is reversible and that the observed reduction in viral titer requires some other explanation.     

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate ($\text{P}\text{Rib}\text{-PP}$).We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by $\text{P}\text{Rib}\text{-PP}$. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate $\text{P}\text{Rib}\text{-PP}$-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine.     

Incorporation of teratocarcinoma stem cells into blastocysts by aggregation with cleavage-stage embryos

Embryonal carcinoma cells from the in vitro teratocarcinoma cell line PSA-1 were combined with normal, eight-cell stage, embryonic cells of the strain $\text{SWR}\text{J}$. The aggregates compacted and formed apparently normal blastocysts within 48 hr. Glucose phosphate isomerase (GPI) assays of the blastocysts revealed the presence of both PSA-1 and $\text{SWR}\text{J}$ GPI isozymes. Inner cell masses isolated from the blastocysts by immunosurgery expressed predominantly the PSA-1 GPI type.     

Freeze-fracture study on the erythrocyte membrane during malarial parasite invasion

A new method is presented for the quantitative analysis of intercellular adhesive specificity. In this assay, two cell types are mixed, one unlabeled and the other labeled with the fluorescent dye, fluorescamine [4-phenylspiro(feran-2[3H],1'-phthalan)-3,3'-dione]. The resulting aggregates are analyzed by fluorescence microscopy to determine the number of labeled and unlabeled cells per aggregate. Random (nonspecific) aggregation was characterized by a binomial distribution, and adhesive specificity was accordingly quantified by the deviation (as determined by a chi-square test) from the calculated binomial distribution. The labeling procedure was simple and rapid, and experiments with 18 different cell types showed that it did not affect cell viability, morphology, rate and extent of adhesion, plating efficiency, and the capability of myogenic cells to undergo terminal differentiation. Most important, assays with morphologically identifiable cell pairs indicated that the fluorescent label neither induced apparent nor destroyed existing adhesive specificity. The most pronounced adhesive specificities were observed with freshly explanted cells from adult tissues and also with mixtures of simian virus 40-transformed and nontransformed BALB/c 3T3 cells. A glucosamine-6-phosphate N-acetylase-deficient mutant 3T3 line (AD6), however, aggregated randomly with parental 3T3 cells. Lectin-resistant mutant Chinese hamster ovary (CHO) cells displayed marginal adhesive specificity when mixed with normal CHO cells.     

The transition from embryonic to adult isozyme expression in reaggregating cell cultures of mouse brain

The transition from embryonic to adult l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was examined in reaggregating cell cultures of mouse brain. Culture conditions were selected that enabled viable aggregates to be produced from neural cells of mice that ranged in age from the 12th day of gestation to 6 days of postnatal age. In all cultures, an increase in the specific and total activity of l-glycerol 3-phosphate dehydrogenase was observed and this was due to an increase in the adult isozyme. The cultures from the older cells, i.e., cerebellar aggregates from mice 6 days of age, expressed approximately a 50-fold increase in activity, whereas cerebellar aggregates from newborn mice exhibited about a three-fold increase in activity. In both cases, the time course for the developmental increase followed the normal temporal sequence. The amount of l-glycerol 3-phosphate dehydrogenase activity in a cerebellar aggregate from a 6-day-old mouse was greater than 1% of the soluble cellular protein, which is about seven times higher than the specific activity determined in the cerebellums of adult mice. Alleles at the structural locus for l-glycerol 3-phosphate dehydrogenase, the Gdc-1 locus, and those alleles that control the activity levels in the cerebellums of mice were expressed in aggregates from $\text{BALB}\text{cBy}$ and $\text{C57BL}\text{6J}$ neonates. However, although the expected structural allele was expressed in all cultures, the expression of activity differences in cerebellar aggregates depended on the age of the mouse from which the cerebellar cells were isolated. Acetylcholinesterase (EC 3.1.1.7) was also measured in these aggregates; the specific activities were highest in aggregates from mice in the 16th day of gestation and least in the cerebellar aggregates of neonates, a trend that was opposite to that of l-glycerol 3-phosphate dehydrogenase.     

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.     

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts

Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+- independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.     

Assay of intercellular adhesiveness using cell-coated Sephadex beads as collecting particles.

A simple, rapid and precise method, based on a previous method, for measuring relative rates of intercellular adhesion is described. DEAE-Sephadex beads were treated with nitrocellulose in order to allow cells to grow on their surfaces. Balb/c 3T3 and Balb/c 3T12 cells were used to characterize the assay. They formed confluent cell layers on nitrocellulose-treated DEAE-Sephadex. These cell-coated beads were employed to collect 32P-labelled cells from single cell suspensions. Since they formed statistically uniform, large collecting surfaces, the collection of labelled cells was markedly improved as compared to the original assay. The cell-coated beads collected a large percentage of the labelled cells in a short time. The percentage of cells collected was independent of the concentration of labelled cells in the assay mixture, and the collection was linear for approximately 60 min. The variability between replicate assays was usually +/- 5%. The assay allows the rapid and precise determination of intercellular adhesion in large numbers of individual samples. These features make it useful to screen for effects of different treatments on intercellular adhesions.     

Cytotoxicity of pyocin S2 to tumor and normal cells and its interaction with cell surfaces

Cytotoxicity and adsorption of pyocin S2 produced by Pseudomonas aeruginosa M47 (PAO 3047) to virally transformed mammalian cells, human malignant cells and normal cells in the same species were studied. Pyocin S2 inhibited the growth of not only tumor cells (XC, TSV-5, mKS-A TU-7, HeLa-S3 and AS-II cells) but also normal cells (BALB/3T3 and BHK 21 cells). The inhibitory effects on the cells increased with an increase of pyocin S2 activity. On the other hand, there were some tumor cells (155-4 T2 and HGC-27 cells) and normal cells (normal rat kidney and human embryo lung cells) which were resistant to pyocin S2. The pyosin S2 activity was neutralized by the cell membrane preparations from pyosin S2-sensitive cells, but not by those from pyocin-resistant cells. This neutralization ability was inhibited by high concentrations of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine and}\text{N-}\text{acetyl}$ neuraminic acid and completely destroyed by periodate and neuraminidase. The inhibition by the saccharides was concentration dependent. These results suggest that the toxicity of pyocin S2 to several mammalian cells is due to the presence of the binding site for pyocin S2 in the cell membrane and further, that the carbohydrate moiety, especially of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine}$ and sialic acid, may play an important role as an initial binding site for pyocin S2.     

Ultrastructure and surface topography of aggregates of human limb mesenchymal cells (HLM15) in vitro.

Tissue-like aggregates of human embryo fibroblasts can be created in vitro by limited aspiration of cells released from substrate during subcultivation. Aggregates increase in size, exhibit intercellular junctions, display a surface topography characteristic of cellular movement, elaborate an extracellular matrix and possess features of cellular death and phagocytosis. These cells, when introduced to a new culture environment, do not migrate away from one another as is common when a primary culture is started from tissue fragments. Instead, cells exhibit continued contact with each other, and develop complex junctional structures during that association. Cellular aggregates generated in this manner may provide a useful system for providing further information on cellular adhesion, intercellular communication, morphogenetic cell movements and the mechanisms of cell death.     

Intercellular adhesive selectivity. I. An improved assay for the measurement of embryonic chick intercellular adhesion (liver and other tissues)

An improved assay for measuring intercellular adhesive selectivity of embryonic chick liver cells is described. Three major improvements over earlier procedures are noted: (a) enhanced reproducibility of liver cell-liver cell aggregate adhesion (homotypic adhesion) was achieved; (b) 25-70% of the input cells adhered to the collecting aggregates during the course of routine experiments as compared to the 0.25% in earlier assays. This increase in cellular adhesion suggests that the observed cell pick-up is a characteristic of the majority of the dissociated liver cell population; (c) the rate of intercellular adhesion was increased 1,000-fold. The main feature of the assay is that it measures the tissue adhesive selectivities of the dissociated cell population. Studies were undertaken on three embryonic chick tissues (liver, neural retina, and mesencephalon) to determine the tissue selectivity of intercellular adhesion of these dissociated cell types. Some general properties of liver cell homotypic adhesion have been studied and are reported.     

Increased synthesis of L-glycerol 3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells

Reaggregating cell cultures of neonatal mouse cerebellar cells express many of the differentiated properties of normal developing cerebellum, including the transition for the embryonic and adult isozymes of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). In order to determine the mechanism leading to increased levels of adult isozyme, aggregates in culture from 2 to 17 days were labeled with radioactive leucine and the relative rate of enzyme synthesis was measured after purification of the enzyme by affinity chromatography on Blue Sepharose 6B. During the course of in vitro differentiation, the relative rate of synthesis increased 100-fold, such that it represented 0.5% of the total protein synthesized in the cytoplasmic fraction of the cell. In vivo, $\text{BALB}\text{cBy}$ mice have twice the level of enzyme activity in the cerebellum as do $\text{C57BL}\text{6J}$ mice. Reaggregating cell cultures of cerebellar cells from these strains of mice also express a difference in the activity level, but only when the cerebellar cells are taken from mice 4 days of age or less. When the relative rates of synthesis of l-glycerol 3-phosphate dehydrogenase were measured in cultures expressing the strain-dependent difference in activity, these rates were found to be approximately twofold greater in cultures of $\text{BALB}\text{cBy}$ cells. In contrast, estimates of the relative rate of enzyme degradation by the double-isotope labeling technique indicate that neither specific enzyme degradation nor degradation of total protein is different in aggregates from the two strains of mice. The results suggest that the genetic mechanisms controlling the levels of l-glycerol 3-phosphate dehydrogenase in the cerebellum during development are intrinsic to the cells and, with the exception of serum factors, are independent of systemic influences.