Proteolysis by Trypsin or Transformation by Polyoma Virus Potentiates the Manganese-Stimulated Adhesion of Fibroblasts to Serum-Coated Substratum

Bivalent cations, such as CaZ+, Mgz+ or Mnz+, are required for the adhesion of cells t o serum-coated substratum, and MnZ+ appears t o be particularly effective in this respect (Rabinowitch & DeStefano, 1973; Yasuda, 1974). We report here the stimulation by bivalent cations of adhesion of BHK-21/CI 3 cells (BHK) and their polyoma-transformed derivatives (PyBHK) to serum-coated glass by using an adaptation of the assay by Walther et a / . (1973). Serum surfaces were prepared by incubating scintillation vials with 50% (v/v) foetal calf serum overnight. The incubation medium in which the adhesion assay was conducted was based on the Glasgow modification of Eagle's medium without bivalent cations, bicarbonate or phosphate but buffered with 25mhl-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The required bivalent cations were dissolved in this buffer before conducting the assay. Single radioactive cells were prepared by minimal proteolysis, with trypsin (0.05 %)/EDTA (0.02 %), of monolayers of cells grown in ''C-labelled protein hydrolysate. Before use the single radioactive cells were washed three times with Eagle's medium, without bivalent cations, with 10% calf serum in the first wash. After incubation of the cells in the scintillation vials, the media and non-adhering cells were poured off and the surface was washed four times t o remove loosely attached cells. Results of typical experiments (Fig. I ) show that initial adhesion of PyBHK cells to serum was more responsive to MnZf than to Caz+ or Mg2+. This increased effect of Mn2+ was greatest after about 20-30min incubation, when it was double that in 1 mM-MgCI, or I mM-CaCI,. After 3 h incubation, there was always a fall in the number of PyBHK cells adhered to serum in MnZ+ buffer that did not occur with cells in Mgz+ or Caz+ buffer, Interference contrast microscopy showed that cells adhering in MnZ+ buffer differed in two ways from the spreading and fibroblastic morphology shown by cells adhering in Ca2+ or Mgz+ buffer. Firstly, although the PyBHK cells spread rapidly in the first 20min in MnZt buffer, they did so in an uncontrolled manner with protrusions of large areas of ruffles. Secondly, after about 90min in the presence of Mn2+, but not Mg2+ or Ca2+, the PyBHK cells retracted with the resulting loss of some adhering cells after 3 h. Non-transformed BHK cells were less responsive t o the effect of Mn2+ than were the transformed PyBHK cells. BHK cells in MnZ+ did show uncontrolled spreading and ruffling and this was followed by cell retraction, but it was noticeably less than that in PyBHK cells. BHK cells did not show the initial increase in Mnz+-stimulated adhesion over that in Ca2+ and Mg2+ and there was no loss of adhering BHK cells after 4 h in MnZ+ buffer. The adhesion assay (Fig. 1 ) involves the cells resisting a shearing force during the washing procedure. The more firmly spread a cell, the more resistant it is to shear and removal from the substratum. This provides a reasonable explanation for the greater adhesion of PyBHK cells in Mn2+ compared with that in Ca2+ or Mgz+. MnZ+ causes an atypical but intense initial cell spreading that makes the cell more resistant to shear and therefore results in greater adhesion, but the subsequent cell retraction means the the cell is less resistant to shear with loss of adhesion (Fig. I ) . BHK cells are less responsive to MnZf and therefore d o not show the greater initial adhesion in Mn2+ or any loss of this adhesion after 3 h. The rate and extent of adhesion for BHK cells and PyBHK cells in Caz+ or MgZt buffer were the same.