ROLE OF SERUM IN THE MORPHOLOGICAL TRANSFORMATION OF SYRIAN-HAMSTER EMBRYO CELLS - CHARACTERIZATION AND PARTIAL-PURIFICATION OF PROTEIN FACTORS IN FETAL BOVINE SERUM
E. Rivedal et U. Haddeland, ROLE OF SERUM IN THE MORPHOLOGICAL TRANSFORMATION OF SYRIAN-HAMSTER EMBRYO CELLS - CHARACTERIZATION AND PARTIAL-PURIFICATION OF PROTEIN FACTORS IN FETAL BOVINE SERUM, Toxicology in vitro, 10(2), 1996, pp. 217
The expression of transformed colony morphology in Syrian hamster embr
yo (SHE) cells, and thus the results obtained in the SHE cell transfor
mation Essay, Is dependent on the source of the foetal bovine serum (F
BS) 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 a
nd non-dialysable), sensitive to heating and thiol reagents, but resis
tant to acid and solvent treatment. The active factor(s) were found to
bind to a number of protein purification media for ion exchange or af
finity purification, and it was initially difficult to reconstitute th
e biological activity from eluted fractions. This loss of activity was
not caused by the separation of more than one necessary factor, but b
y the factors being highly hydrophobic and negatively charged, and the
refore strongly bound to the column material. The active factors could
be eluted from Affigel Blue in 50% ethylene glycol, but not in 4 M Na
Cl. The bioactive protein fraction could be further fractionated by ge
l permeation chromatography on Biogel P-60 in 1 M acetic acid, and cat
ion exchange chromatography on MonoS with 20% acetonitrile added to th
e 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 e
luted at propanol concentrations of 20 and 27%, respectively. In the p
resent studies, we were unable to identify the factors with transforma
tion supporting activity, probably because of the high content of prot
ein/peptides with similar biochemical properties in FBS. In further st
udies we will seek to demonstrate whether previously isolated growth f
actors, or signalling substances, with similar biochemical properties
support the expression of the morphologically transformed phenotype in
SHE cells. Copyright (C) 1996 Elsevier Science Ltd.