Sorting out of normal and virus-transformed cells in cellular aggregates

The sorting-out behavior (self-segregation of two cell types from mixtures of the two) of five different established cell lines was studied. Eight of the ten possible binary combinations of these lines, cultured as cellular aggregates, were examined. Mouse BALB/c 3T3 cells sorted out internally to the corresponding malignant SV40 virus-transformed 3T3 cells. The transformed 3T3 line (SVT-2) did not sort out from a revertant line selected from SVT-2 cells by resistance to concanavalin A (con A). The revertant cells sorted out externally to the parent BALB/c 3T3 cells, although segregation was generally incomplete. BALB/c 3T3 cells did not sort out from another contact-inhibited line of 3T3 cells derived from Swiss albino mice (Swiss 3T3). Both BALB/c 3T3 and Swiss 3T3 cells sorted out from cells of the contact-inhibited hamster line, NIL B. Instead of a two-layered sphere, however, a three-layered structure was observed with most of the NIL B cells external to the 3T3 cells, and a few NIL B cells comprising the center of the sphere. On the other hand, NIL B cells did not consistently sort out from either the SVT-2 or con A cells. In general, sorting out between pairs of these five lines are slower and less complete than is generally observed between the more extensively studied chick embryonic tissue cells, suggesting that the cultured cells may be more closely related in their adhesive properties. The internal segregation of BALB/c 3T3 cells relative to SVT-2 cells is consistent with the hypothesis that transformed cells are less adhesive than their nontransformed counterparts.     

Chromatin fractionation of normal and virus-transformed cells in culture

A simple technique for the obtaining of purified chromatin fractions from mammalian cells in culture is described. The procedure involves the isolation of clean nuclei in 0.30 M sucrose, 1.5 mM MgCl₂, 0.2 mM CaCl₂, 0.01 M Tris HCl pH 7.2, followed by sonication and differential centrifugation to collect specific chromatin fractions. Heterochromatin of SV-40 and Rous sarcoma virus transformed 3T3 cells showed a 6- to 7-fold enrichment in satellite DNA while a 2- to 3-fold enrichment in repetitive DNA was obtained in established and RSV transformed cells of Microtus agrestis. This method will facilitate the search for the site of integration of oncogenic viruses in the chromatin of mammalian cells.     

Topography of substrate-attached glycoproteins from normal and virus-transformed cells

The topographical distribution of substrate-attached material (SAM) which may mediate adhesion of BALB/c 3T3 and SV40-transformed 3T3 cells to the culture substrate has been examined at both the macroscopic and microscopic levels. Autoradiographic detection of these ‘glycoproteins’ on the substrate subsequent to incorporation of ¹⁴C-glucosamine or ³⁵S-methionine and exposure to X-ray film indicated:

1. 1. Evenly distributed glucosamine-labeled SAM on the substrate of confluent cultures.

2. 2. A deficiency in the methionine content of SAM from transformed cells.

3. 3. Direct deposition of these materials onto the substrate at cell colony locations, and not randomly on the substrate subsequent to secretion into the medium.

Autoradiographic detection of these ‘glycoproteins’ subsequent to ³H-glucosamine (or ³H-leucine) incorporation and exposure to liquid emulsion indicated:

1. 1. Substrate-attached glycoproteins are present in focal pools on the underside of the cell.

2. 2. These focal pools are distributed comparably, in terms of number of foci per area of substrate, for confluent normal and transformed cells. The patterns of SAM deposited by freshly-attaching and spreading 3T3 cells has also been examined. The evidence suggests that cells are adherent to the substrate at localized areas which appear to be evenly distributed on the underside of normal or transformed cells.

     

Electron spin resonance of growing normal and virus-transformed cells

A g = 2.003 ESR signal, attributed to a free radical localized in HeLa cell nuclei and mitochondria but absent in membranes and cytoplasm, has been studied as a function of the culture growth cycle in normal (NRK and 3T3) and virus transformed (NRK/RSV and 3T3/SV40) cells. For both these cell pairs, the signal is higher during the "lag" stage and lower during the "growth" stage. The average specific intensity of the signal in normal cells is about twice that in virus-transformed cells. However, the maximal point of resonance during the lag state is higher in transformed cells than in normal ones. The lag stage in NRK and NRK/RSV cells is much longer than in 3T3 and 3T3/SV40 cells, while the maximal value of the g = 2.003 ESR signal occurs, early in the lag stage of 3T3 and 3T3/SV40 cells and late in the lag stage of NRK and NRK/RSV cells.     

Diffusion and deformations of single hydra cells in cellular aggregates

Cell motion within cellular aggregates consists of both random and coherent components. We used confocal microscopy to study the center of mass displacements and deformations of single endodermal Hydra cells in two kinds of cellular aggregates, ectodermal and endodermal. We first carefully characterize the center of mass displacements using standard statistical analysis. In both aggregates, cells perform a persistent random walk, with the diffusion constant smaller in the more cohesive endodermal aggregate. We show that a simple parametric method is able to describe cell deformations and relate them to displacements. These deformations are random, with their amplitude and direction uncorrelated with the center of mass motion. Unlike for an isolated cell on a substrate, the random forces exerted by the surrounding cells predominate over the deformation of the cell itself, causing the displacements of a cell within an aggregate.     

Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.     

Poly(A) polymerases in normal and virus-transformed cells.

The presence of poly(A) polymerase(s) has been studied in a clone of the established hamster embryo fibroblast line (NIL), and in a subclone of this line transformed by an RNA tumour virus, hamster sarcoma virus, (NIL-HS VIRUS). The results suggest the existence of three distinct poly(A) polymerases, designated I, IIA and IIB, in dense cultures of NIL and NIL-HS virus cells. Forms IIA and IIB have also been found in exponentially growing NIL and NIL-HS virus cells. Poly(A) polymerase I has not been detected in growing NIL cells, while growing and resting NIL-HS virus contain comparable amounts of this enzyme. The poly(A) polymerases differ in chromatographic behaviour and in their requirements for divalent cations. They are highly specific for ATP and require the presence of a primer. Cellular RNA or poly(A), but not the oligoribonucleotide (Ap)4A, can be utilized as primers. The products of the reactions have been identified as poly(A) chains (20-50 nucleotides long) by alkaline degradation and by their resistance to pancreatic RNAase.     

Cell shape and hexose transport in normal and virus-transformed cells in culture

The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only in the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer.     

Localization and organization of microfilaments and related proteins in normal and virus-transformed cells

The localization and organization of actin-like microfilaments in normal, SV-40 and adenovirus transformed cells are determined by the coordinated use of light optical, electron optical and biochemical techniques. In adenovirus-type 5 transformed hamster embryo cells, microfilament meshworks appear to be the predominant organizational form of cellular actin, while in normal hamster cells, microfilament bundles are prevalent. Differences between 3T3 and SV-40 transformed 3T3 cells are less apparent and may be related to the packing and intracellular distribution of microfilament bundles. Attempts at relating these ultrastructural changes in transformed cells to the images obtained following reaction with fluorescein-labelled myosin fragments and indirect immunofluorescence with smooth muscle myosin antibody are discussed. In several instances the fluorescence microscope images do not correspond to the ultrastructural observations. The results are discussed in terms of the possible relationships between alterations in cytoplasmic contractile elements and the abnormal behavior of transformed cells.     

Metabolic properties of substrate-attached glycoproteins from normal and virus-transformed cells.

Balb/c 3T3, SV40-transformed 3T3 (SVT2), and Con A revertant variants of transformed cells leave a layer of glycoprotein on the culture substrate upon EGTA mediated removal of cells. The metabolic properties of this substrate-attached material (glycoprotein) have been examined. Pulse and cumulative radiolabeling experiments with glucosamine and leucine precursors established that this substrate-attached material accumulates on the substrate in growing cultures until cells have completely covered the substrate. The synthesis and/or deposition of the material diminished dramatically in cultures whose substrates had been completely covered with cells as observed microscopically, even though the contact-inhibited cell lines continued to make cell-associated and medium-secreted glycoproteins and transformed cells continued to divide and form multilayered cultures. Pulse-chase analysis using long periods of pulsing with radioactive leucine demonstrated that these glycoproteins are deposited directly on the substrate by cells and not subsequent to secretion into the medium. The substrate-attached material accumulated during long pulses was stably adherent to the substrate and displayed little appreciable turnover during 3 days of chasing of either sparse or dense cultures. Short-term pulse-chase analysis with leucine revealed two metabolically different pools of material-one which turns over very rapidly with a half-life of 2-3 hr (observed in both low-density and high-density cultures) and a second pool which is stably deposited on the substrate and whose proportion increased with the length of the radiolabeling period. No appreciable differences in the metabolic properties of substrate-attached material were observed in the three cell types studied during growth on a plastic substrate. These results are discussed with regard to the implicated roles of these glycoproteins in in mediating adhesion of normal and virus-transformed cells to the substrate.     

In vitro methylation of total and foldback DNAs in normal and virus-transformed cells

The levels of the in vitro methylation of total and palindromic DNAs in nuclei isolated from normal and virus-transformed cells are compared. The methylation rate of total DNA in normal rat kidney cells is much higher than that detected in normal mouse fibroblasts. However, for both cell species, while the maximal rate of DNA methylation is observed in the mid-logarithmic phase of the cell culture growth, palindromes are always found to be more heavily methylated than total DNA. The 5-methylcytosine content of DNA, especially of palindromes, is higher in virus-transformed cells than in untransformed cells.     

Electrophoretic analysis of substrate-attached proteins from normal and virus-transformed cells.

The proteins which have been left tightly bound to the tissue culture substrate after ethylenebis (oxyethyl-enenitrilo) tetraacetic acid (EGTA)-mediated removal of normal, virus-transformed, and revertant mouse cells and which have been implicated in the substrate adhesion process have been analyzed by slab sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three size classes of hyaluronate proteoglycans were resolved in the 5% well gel; approximately half of the protein in the substrate-attached material coelectrophoresed with these polysaccharides-so-called glycosaminoglycan-associated protein(GAP). A portion of the GAP was shown to be highly heterogeneous and displaced from the polysaccharide by preincubation with calf histone before electrophoresis. The relative proportions of the proteoglycans varied in material deposited during a variety of cellular attachment and growth conditions. The remainder of the cellular protein in substrate-attached material was resolved as several major and distinct protein bands in 8 or 20% separating gels (a limited number of distinct serum proteins have also been identified as substrate bound). Protein C0 (molecular weight 220 000) was a prominent component in the material from a variety of normal and virus-transformed cells and resembled the so-called LETS or CSP glycoprotein in several respects; protein Ca was myosin-like in several respects; protein C2 was shown to be actin; and protein C1 (molecular weight 56 000) does not appear to be tubulin. Histones were also present in most preparations of substrate-attached material, particularly at high levels in transformed cell meterial, and may result from EGTA-mediated leakiness of the cell and subsequent binding to the negatively charged polysaccharide. These substrate-attached proteins were (a) prominent in substrate-attached material from many cell types in characteristic relative proportions, (b) deposited by EGTA-subcultured cells during the first hour of attachment to fresh substrate, (c) deposited by cells growing on plastic or glass substrates (three additional) components were also prominent in glass-attached material), and (d) deposited during long-term growth on or initial attachment to substrates coated wit 3T3 substrate-attached material. Pulse-chase analyses with radioactive leucine indicated that these proteins exhibit different turn-over behaviors. These results are discussed with regard to the possible involvement of these substrate-attached proteins in the substrate adhesion process, with particular interest in the interaction of cytoskeletal microfilaments with other surface membrane components and with regard to alteration of substrate adhesion by virus transformation.     

Molecular composition and origin of substrate-attached material from normal and virus-transformed cells

The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein C_o (the LETS glycoprotein); protein C_a (a myosin-like protein); protein C_b (MW 85,000); protein C₁ (MW 56,000, which is apparently not tubulin); protein C₂ (actin); proteins C₃–C₅ (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins C_c, C_d, C_e, and C_f. The LETS glycoprotein (C_o) and C_d appear in newly-synthesized SAM (which is probably enriched in “footpad” material – “footpads” being focal areas of subsurface membranous contact with the substrate) in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in “footprint” material – remnants of footpads left behind as cells move across the substrate). C_o and C_d turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell “surface” complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of “footpad” vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.