Application of the principles of enzyme kinetics to clonal growth rate assays: an approach for delineating interactions among growth promoting agents.

The interaction of mitogenic factors on a single cell type and the comparative activity of a given factor in diverse cell types have been studied by applying the principles of Michaelis-Menten kinetics to clonal growth data. Such comparisons are facilitated by derivation of two parameters; Km mitogen, the mitogen concentration that gives half-maximal clonal growth and a theoretical maximal growth rate, RMAX T. Both parameters are analogous to the Km and VMAX as applied to enzymatic reactions. Use of these parameters permits meaningful comparisons between cells with different growth rates. Using kinetic analysis of dose-response data, we found that normal human epithelial cells require 200 times more fetal bovine serum protein (FBSP) than a malignant line to multiply at their respective half-maximal rates. Further, the Km FBSP of normal cells was reduced to that of the malignant line by the inclusion of growth factors (EGF or FGF, and hydrocortisone) in the medium. On the other hand, even though greater levels of serum were required when growth factors and hydrocortisone were not present, their inclusion did not alter RMAX T. Interactions between mitogenic factors were shown to be unidirectional. Although EGF reduced the Km FBSP, FBSP did not change the Km EGF. The same type of analysis revealed that hydrocortisone, which potentiated the mitogenic activity of EGF did not change the Km EGF. Kinetic analysis of cell growth should prove useful in studies on the relation between growth and tumor promotion as well as in the evaluation of growth-inhibiting chemotherapeutic agents.