Proposed role of phosphatidic acid in the extracellular control of the transition from G2 phase to mitosis exerted by epidermal growth factor in A431 cells.

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)