Epidermal growth factor (
EGF) has been shown to cause an inhibition of A431 cells in G2 phase within approximately 10 min, i.e., shortly before mitosis (Kinzel et al.,
Cancer Res., 50: 7932-7936, 1990). This system has been used to study the proposed role
phospholipid metabolites, particularly
phosphatidic acid (PA), may play (Kaszkin et al.,
Cancer Res., 51: 4328-4335, 1991) in the extracellular control of cells at the physiological restriction site in G2 phase. A431 cells responded to
EGF with a dose-dependent formation of
phosphatidic acid (PA) which correlated with the dose-dependent G2 delay as well as with their time courses. The G2 delay induced by
EGF as well as PA mobilization were effected in
conditioned medium or in fresh medium containing bovine serum albimun instead of serum, i.e., under the conditions necessary for precursor studies to be carried out. The major pathway of PA formation was probably via
phospholipase C-mediated breakdown of
phosphatidylinositol and
diacylglycerol kinase: (a) the dose response of PA formation correlated with that of total
inositol phosphate accumulation; (b) little
diacylglycerol was found and then only at a high
EGF concentration; (c) prelabeling with [1-14C]
arachidonic acid resulting in a large specific labeling of
phosphatidylinositol led to an
EGF-induced, dose-dependent formation of radioactive arachidonyl-PA (correlated with that of total PA and
inositol phosphate), but in the presence of a primary alcohol not to the formation of radioactive phosphatidylalcohol; (d) prelabeling with [1-14C]
oleic acid led to the
EGF-induced formation of labeled PA, which in the presence of a primary alcohol was only slightly reduced to the advantage of very low levels of labeled phosphatidyl alcohol, thus demonstrating that an
EGF-effected activation of
phospholipase D did occur but contributed little to the general PA level. An alternative mobilization of PA was attempted with the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA), which was shown to activate
phospholipase D in A431 cells and to elicit PA from a
phospholipid pool which was not significantly labeled with radioactive
arachidonic acid. The TPA-induced degree of PA formation and of the G2 delay correlated. Both phenomena were considerably larger with fresh medium containing 0.5%
bovine serum albumin instead of serum than in
conditioned medium.(ABSTRACT TRUNCATED AT 400 WORDS)