Cell attachment to a substratum and cell surface proteases

The attachment of BHK cells to a substratum in serum-containing medium was inhibited when inhibitors of proteolytic enzymes were added to the incubation medium. The inhibitors found to be effective were l-1-tosylamide-2-phenylethyl chloromethyl ketone, N-α-p-tosyl-l-lysine chloromethyl ketone HCl, and phenylmethyl sulfonyl fluoride. Soybean trypsin inhibitor had no effect on cell attachment when added to the incubation medium. Comparative studies were carried out with normal, virus-transformed and suspension-culture adapted BHK cells in which the cells were treated with various concentrations of l-1-tosylamide-2-phenylethyl chloromethyl ketone and then resuspended in fresh medium and tested for their adhesiveness. The relative efficiency of inhibition by l-1-tosylamide-2-phenylethyl chloromethyl ketone was similar with all three cell lines. The possibility was investigated that cell attachment involves proteolytic activation by cell surface proteases of serum proteins absorbed to the substratum. It was found that trypsin or chymotrypsin treatment of substrata coated with serum resulted in an enhanced rate of subsequent cell attachment to the substrata. This effect did not involve bulk removal of serum from the substrata or the presence of residual trypsin in the incubations. Moreover, inhibition of cell attachment by l-1-tosylamide-2-phenylethyl chloromethyl ketone was less pronounced when cell attachment to trypsin-activated substrata was compared with attachment to unactivated substrata. In related studies, it was observed that the spreading of BHK cells onto their substratum requires the presence of serum components in solution in the incubation medium.