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

Abstract

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.

Fibronectin and proteoglycans as determinants of cell‐substratum adhesion

“…Fibronectin shows a similarly restricted distribution when visualized by indirect immunofluorescence (see below). Furthermore, deposition of radiolabeled SAM onto the substratum ceases when the cells have completely covered the substratum, although the amount of radiolabeled macromolecular material shed into the medium continues to increase [19]. At least some of this material should have access t o the substratum, since antibody is able to penetrate under the cells (see below).…”

Requirements for Brain Cell Attachment, Survival and Growth in Serum-Free Medium: Effects of Extracellular Matrix, Epidermal Growth Factor and Fibroblast Growth Factor

“…He proposed that the dif ference between cells requiring or not requir ing serum for spreading might depend upon whether the cells were able to produce an ap propriate spreading factor (o coat the sub strata. since Culp et al [1975] had described a glycoprotein which was secreted by a fibro blast cell-line and adsorbed to the sub stratum. In his review, Grinnell [1978] consi dered the role of microexudates and con ditioned medium in cell adhesion.…”

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

“…A variety of experimental approaches have now been used in our laboratory to indicate that most of the components in SAM may be direct participants in the cell-substrate adhesion process, although definitive proof for their precise molecular role is still lacking. Pulse-chase analysis of the metabolic source of SAM (8) and autoradiography experiments to determine its topographical relationship with regard to the location and movement of cells on the substrate (9) indicated that SAM had not resulted from secretion of components into the medium followed by nonspecific binding to the substrate, but that SAM was some portion of the cellular "footpads" on the underside surface by which the cell adheres to the substrate (10, 11).…”

“…"Footpads" (10,11,28) are localized blebs of surface membrane on the underside surface of the cell by which it adheres to the substrate, and whose distribution on the substrate is similar to that of glucosamine-or leucine-radiolabeled SAM detected autoradiographically (9). ality between increased cell growth and increased accumulation of SAM (either glucosamine-or leucine-radiolabeled) until the substrate surface was completely covered with cells (8). At that point, SAM accumulation discontinued, even though transformed cells continued to grow and pile into dense layers.…”

“…Culp et al (8) reported that SAM consisted of 2 different metabolic pools of proteins: a major portion of the protein radiolabeled over long periods of cell growth was stably bound to the substrate, while a pool of protein appearing in SAM during short radiolabeling periods turns over with a half-life of 2-4 hours. The kinetics of turnover of this latter pool of protein are very similar to the kinetics of turnover of the small amount of sulfated-GAG found in SAM (1 3).…”