Mammalian cell growth proteins. I. Growth stimulation of fetuin.

In earlier papers we described the presence and properties of a factor in the fetuin fraction of fetal calf serum which promotes growth of mammalian cells in vitro and enhances their stretching on glass and other surfaces.1 Fetuin is an alpha-globulin, obtained by fractional precipitation with ammonium sulfate, whose major component is a glycoprotein of molecular weight 45,000.1 A synthetic medium was described which when supplemented with both albumin and fetuin produces virtually 100 per cent efficiency of colony growth of single HeLa cells plated in vitro.' Another synthetic medium, F12,2 was later developed in this laboratory which eliminated the need for albumin, greatly reduced the amount of fetuin required for maximal cell growth, and supported slow growth of some but not all cell strains without any protein supplement. Other laboratories have found or confirmed the presence of a growth-stimulating, alpha-globulin serum component,3 but questions have been raised as to whether the active fraction of fetal serum is indeed fetuin, and whether growth-promoting proteins from adult sera are similar to or different from those found in fetal sera.38' 4 The divergences of interpretation which have arisen are due to a variety of causes, including the presence of several components3 in the original fetuin described by Pedersen;6 the ease with which active fetuin preparations can lose their biological activity;" the lack of reliable, quantitative tests for titration of the biological activity of this growth-promoting factor; and the fact that various investigators have used different cells, media, and methodologies for assessment of the biological activities of their fractions. The present paper attempts to clarify further the relationship between the various materials that may be present in the fetuin fraction of fetal calf serum and the biological activity observed for the fraction as a whole. Methods.-Cells: Three types of cells were used in these experiments: a standard strain of bovine ovary tissue culture cells isolated in this laboratory in 1966, the Chinese hamster ovary cell,8 and the mouse L cell. Cells were farmed in F12 supplemented with 10%0 fetal calf serum by methods described previously.7 Single cell platings were made in plastic Petri dishes (Falcon) as previously described.7 Plating efficiencies, i.e., the fraction of single cells plated which develop into countable colonies, usually reached 75-100% in the presence of 0.50% or more of fetal calf serum which was always present in a set of control plates in each experiment. Plating efficiencies in test plates were routinely expressed as a fraction of the values obtained on the control plates. Fetuin: Standard fetuin was prepared by(NH4)2S04 fractionation following the procedure of Fisher,9 which was adapted from the Pedersen method,6 but gives a higher yield of material with no sacrifice of specific biologic activity. It contains a small amount of albumin and of a high-molecular-weight component which has been regarded as a polymer of the major component.5 In the experiments on more highly purified fetuin, the standard fetuin (which had already received four successive precipitations in 50% saturated (NH4)2S04 alternating with solubilization in saline, followed by water dialysis and filtration to eliminate euglobulins) was made the starting point for further purification. Three