Ionizing radiation stimulates existing signal transduction pathways involving the activation of epidermal growth factor receptor and ERBB-3, and changes of intracellular calcium in A431 human squamous carcinoma cells

Previous studies demonstrated that ionizing radiation activates the epiderm al growth factor receptor (EGFR), as measured by Tyr autophosphorylation, a nd induces transient increases in cytosolic free [Ca2+], [Ca2+](f). The mec hanistic linkage between these events has been investigated in A431 squamou s carcinoma cells with the EGFR Tyr kinase inhibitor, AG1478. EGFR autophos phorylation induced by radiation at doses of 0.5-5 Gy or EGF concentrations of 1-10 ng/ml is inhibited by > 75% at 100 nM AG1478. Activation of EGFR e nhances IP3 production as a result of phospholipase C (PLC) activation. At the doses used, radiation stimulates Tyr phosphorylation of both, PLC gamma and erbB-3, and also mediates the association between erbB-3 and PLC gamma not previously described. The increased erbB-3 Tyr phosphorylation is to a significant extent due to transactivation by EGFR as > 70% of radiation- a nd EGF-induced erbB-3 Tyr phosphorylation is inhibited by AG1478. The radia tion-induced changes in [Ca2+](f) are dependent upon EGFR, erbB-3 and PLC g amma activation since radiation stimulated IP3 formation and Ca2+ oscillati ons are inhibited by AG1478, the PLC gamma inhibitor U73122 or neutralizing antibody against an extracellular epitope of erbB-3. These results demonst rate that radiation induces qualitatively and quantitatively similar respon ses to EGF in stimulation of the plasma membrane-associated receptor Tyr ki nases and immediate downstream effecters, such as PLC gamma and Ca2+.