Epidermal growth factor-induced centrosomal separation: mechanism and relationship to mitogenesis.

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Abstract
Using a rabbit antibody to MAP1 [microtubule associated protein] to stain centrosomes the mechanism by which epidermal growth factor (EGF) induces centrosomal separation in HeLa [human cervical carcinoma] cells was studied. The response is rapid, being detectable within 20 min after EGF (100 ng/ml) addition and by 4 h 40% of logarithmically growing cells and > 70% of cells synchronized at G1/S with 1 mM hydroxyurea show centrosomes separated by more than one diameter. A concentration of 0.05 ng/ml and 0.1 to 0.5 ng/ml of EGF induces significant separation in synchronized cells (5-9% control vs. 20% with EGF at 0.5 ng/ml induces a half maximal response. Centrosomal separation is blocked by energy inhibitors, trifluoperazine, chlorpromazine and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene Sulfonamide], cytochalasins B and D and taxol, and is stimulated or enhanced by A23187 [calcimycin], colchicine and oncodazole. Trifluoperazine, W-7, cytochalasin D, and taxol also block DNA synthesis in response to EGF as measured by autoradiography using [3H]thymidine. The hypothesis based upon these results is that EGF, by raising the free Ca level, activates calmodulin, which stimulates contraction of microfilaments attached to the centrosome, pulling the daughter centrosome apart. EGF may also induce depolymerization or detachment of microtubules in the vicinity of the centrosome which ordinarily serve to maintain its position and inhibit separation. Centrosomal separation may be a key event in triggering DNA synthesis in response to EGF and colchicine.