Abstract
We have investigated factors controlling the rate of adhesion of suspension culture CHO cells to serum coated glass surfaces. Thus, we have examined (1) the effect of metabolic depletion via KCN treatment; (2) effects of colchicine and cytochalasin‐B, two drugs which are presumptive inhibitors of microtubule and microfilament activities, respectively; (3) effects of temperature on adhesion; (4) the correlation between changes in cell adhesion rate and alterations of lacteroperoxidase labelled cell surface proteins consequent to treatment with proteases.
We have found that colchicine is completely without effect on the initial adhesion process in CHO cells, indicating that microtubules are not a determinant of adhesion kinetics. Cytochalasin‐B and depletion of ATP stores by KCN both diminish CHO cell adhesion, but seem to act in different ways. Treatment with cytochalasin‐B results in a dose dependent reduction in the initial slope of adhesion versus time curves, while KCN treatment produces a pronounced lag period during which little cell attachment takes place, followed by a relatively rapid rise to control levels of cell attachment.
Analysis of CHO cell adhesion kinetics as a function of temperature reveals that the inhibition produced by low temperature seems similar to that produced by KCN. Thus at elevated (25°C) temperature, CHO cells adhere rapidly, while at 4°C adhesion is completely inhibited. However, at intermediate temperatures (10°C) a pronounced lag period followed by a rapid rise is noted, a result similar to that seen in KCN treated cells.
Treatment of CHO cells with low doses (10 μg/ml) of crystalline trypsin results in the loss of several major cell surface proteins, without any appreciable effect on adhesion kinetics. High doses (100–1,000 μg/ml) of trypsin result in the cleavage of other proteins whose loss may be related to a parallel loss in adhesive ability. Some of the CHO cell surface proteins seem resistant to doses of trypsin (1,000 μg/ml) which completely abolish the ability of cells to adhere.
Our observations on the effects of inhibitors, temperature, and surface proteolysis on CHO cell adhesion kinetics suggest the involvement of (a) microfilaments, (b) plasma membrane fluidity, and (c) a subclass of the surface proteins as important determinants of the adhesion process.