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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cell to substratum adhesion-promoting activity released by normal and virus-transformed cells in culture

It is demonstrated here that cultured fibroblasts release into their medium a nondialyzable, protease-sensitive factor(s) capable of promoting the adhesion and spreading of virus-transformed rat fibroblasts on a plastic substratum. A relatively sensitive biological assay is described for quantitation of the adhesion-promoting factor (APF) activity in serum-free, conditioned medium harvested from the cultures. Evidence is presented which indicates that the primary mode of action of the APF is by binding to and modifying the properties of the substratum. Conditioned media harvested after 24 h of incubation in similarly populated cultures of normal fibroblasts of diverse animal species exhibited similar levels of APF activity. However, conditioned media obtained from Rous sarcoma virus (Prague strain)-transformed and avian sarcoma virus B77-transformed rat fibroblasts exhibited three- to sixfold lower levels of APF activity than media conditioned in parallel cultures of heterologous or homologous normal fibroblasts. Cultivation of B77 virus-transformed rat cells in the presence of dibutyryl cyclic AMP and theophylline led to as much as a sevenfold increase in the level of APF activity appearing in the culture medium, with a concomitant increase in the adhesiveness of the cells to the culture substratum. The results support the role of extracellular macromolecules in cell to substratum adhesion. It is postulated that the reduced adhesiveness of transformed cells to a substratum may be at least partially owing to a deficiency in the production and/or release of APF-like macromolecules.     

Histone and nonhistone proteins from normal and virus-transformed rat thyroid epithelial cells

The histone and nonhistone nuclear proteins extracted by different methods from nuclei of normal and virus-transformed rat thyroid epithelial cells (FRTL5 cell line) were studied by polyacrylamide gel electrophoresis in acetic acid/urea and in SDS and by autoradiography. The results have shown that some of these proteins have an higher level of phosphorylation in virus-transformed than in normal cells; moreover, an higher amount of three proteins (C, D, and E), which in normal cells are not detectable at least as Coomassie staining, was found. These proteins, extractable with perchloric acid, are suggested to belong to the High Mobility Group (HMG proteins) and to play some regulatory role.     

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.