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

Abstract

Previous studies demonstrated that ionizing radiation activates the epidermal growth factor receptor (EGFR), as measured by Tyr autophosphorylation, and induces transient increases in cytosolic free [Ca2+], [Ca2+]f. The mechanistic linkage between these events has been investigated in A431 squamous carcinoma cells with the EGFR Tyr kinase inhibitor, AG1478. EGFR autophosphorylation 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 enhances IP3 production as a result of phospholipase C (PLC) activation. At the doses used, radiation stimulates Tyr phosphorylation of both, PLCgamma and erbB-3, and also mediates the association between erbB-3 and PLCgamma not previously described. The increased erbB-3 Tyr phosphorylation is to a significant extent due to transactivation by EGFR as >70% of radiation- and EGF-induced erbB-3 Tyr phosphorylation is inhibited by AG 1478. The radiation-induced changes in [Ca2+]f are dependent upon EGFR, erbB-3 and PLCgamma activation since radiation stimulated IP3 formation and Ca2+ oscillations are inhibited by AG1478, the PLCgamma inhibitor U73122 or neutralizing antibody against an extracellular epitope of erbB-3. These results demonstrate that radiation induces qualitatively and quantitatively similar responses to EGF in stimulation of the plasma membrane-associated receptor Tyr kinases and immediate downstream effectors, such as PLCgamma and Ca2+.