Comparative studies on the carbohydrate-containing membrane components of normal and adenosine 3′:5′-cyclic monophosphate-treated Chinese-hamster ovary cells

Abstract

1. We investigated some of the changes in plasma-membrane composition that accompany the alteration in cell growth and morphology induced by treating Chinese-hamster ovary cells with dibutyryladenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP). 2. A double-labelling technique was employed in which normal cells were given ¹⁴C-labelled precursor, and those treated with dibutyryl cyclic AMP were given ³H label. l-Leucine, d-glucosamine, and l-fucose were used to label the membranes. 3. After 3 days growth, the two populations of cells were harvested by trypsin treatment, the cells were pooled, and plasma membranes isolated. Proteins and glycoproteins of the membranes were separated by electrophoresis on sodium dodecyl sulphate–polyacrylamide gels, and the radioactive profiles for ¹⁴C and ³H and the staining patterns with Amido Black were compared. 4. Although certain components of the membrane from treated cells showed marked quantitative changes, there was neither major addition nor major deletions of components. 5. Complete proteolysis of the mixed membranes, of the material released from the cell surface by trypsin, and of the glycoproteins released from the cells into the medium, gave a series of radioactive glycopeptides when either fucose or glucosamine was employed as precursor. 6. After such glycopeptides were fractionated on columns of Sephadex G-50, marked differences in the elution profiles of ³H and ¹⁴C were noted. Dibutyryl cyclic AMP evidently causes alterations in the overall composition of the carbohydrate components of the cell surface. It was not possible to decide whether this was solely the result of the same glycoproteins being formed but in different proportions, or the result of modifications of oligosaccharide side chains on some of the glycoproteins. 7. Some of the changes were not unlike the reverse of those that accompany the transformation of fibroblasts by oncogenic viruses, and our results lend credence to the idea that the lowered amount of cyclic AMP noted in transformed cells is responsible for their altered surface properties.