Role of serum in the morphological transformation of Syrian hamster embryo cells: Characterization and partial purification of protein factors in foetal bovine serum

Abstract

The expression of transformed colony morphology in Syrian hamster embryo (SHE) cells, and thus the results obtained in the SHE cell transformation assay, is dependent on the source of the foetal bovine serum (FBS) used. The purpose of this study was to characterize the factors in FBS that are necessary for the expression of transformed morphology. The factors were of protein nature (precipitated by ammonium sulfate and non-dialysable), sensitive to heating and thiol reagents, but resistant to acid and solvent treatment. The active factor(s) were found to bind to a number of protein purification media for ion exchange or affinity purification, and it was initially difficult to reconstitute the biological activity from eluted fractions. This loss of activity was not caused by the separation of more than one necessary factor, but by the factors being highly hydrophobic and negatively charged, and therefore strongly bound to the column material. The active factors could be eluted from Affigel Blue in 50% ethylene glycol, but not in 4 m NaCl. The bioactive protein fraction could be further fractionated by gel permeation chromatography on Biogel P-60 in 1 m acetic acid, and cation exchange chromatography on MonoS with 20% acetonitrile added to the buffers. Isoelectric focusing on a Rotofor cell indicated two peaks of transforming activity, one with isoelectric point at about pH 8.5, and one at pH 9.5. The finding of two peaks of biological activity is supported by reversed phase chromatography studies. Bioactivity of two fractions from isoelectric focusing with pI around 8.5 and 9.5 were eluted at propanol concentrations of 20 and 27%, respectively. In the present studies, we were unable to identify the factors with transformation supporting activity, probably because of the high content of protein/peptides with similar biochemical properties in FBS. In further studies we will seek to demonstrate whether previously isolated growth factors, or signalling substances, with similar biochemical properties support the expression of the morphologically transformed phenotype in SHE cells.