Epidermal growth factor receptor, c-erbB2 and c-erbB3 receptor interaction, and related cell cycle kinetics of SK-BR-3 and BT474 breast carcinoma cells

Background: Receptors belonging to the epidermal growth factor receptor (EG FR) family transfer extracellular signals by homotypic and heterotypic rece ptor interaction and cross-activation. Cell differentiation, death, and pro liferation are regulated via these receptor-tyrosine-kinases. However, the initial mechanisms that lead to signal specificity and diversity, which cau se a defined cellular response, are incompletely understood. We investigate d the recruitment of receptor complexes in two c-erbB2-overexpressing breas t carcinoma cell lines, SK-BR-3 and BT474, after ligand binding and its eff ects on intracellular signal transduction and cell cycle regulation. Methods: In order to analyze the coaggregation of receptors on the cell sur face induced by specific growth factor treatment, we used the flow cytometr ic Foerster-type fluorescence resonance energy transfer (FRET) technique. C ell cycle kinetics were monitored flow cytometrically via the anti-BrdU tec hnique and acitivation of intracellular signal cascades was analyzed by Wes tern blotting. Results: After stimulation with EGF BT474, but not SKBR-3, cells formed EGF R/c-erbB2 receptor complexes. Neither EGF nor heregulin (HRG) induced c-erb B2/c-erbB3 receptor complexes in BT474. However, SK-BR-3 cells exhibited a high amount of c-erbB2/c-erbB3 heterodimers even without growth factor stim ulation which could be elevated after prolonged EGF and HRG treatment. In b oth cell lines, mitogen-activated protein kinase (MAPK) phosphorylation was detectable after short-term and prolonged EGF and HRG treatment. However, only SK-BR-3 cells showed a constitutive activation of both protein kinase B (PKB)/Akt and MAPK signaling pathways. Growth factor treatment caused an amplified PKB/Akt activation in this cell line. The induction of EGFR/c-erb B2 complexes in BT474 was associated with shortening of the GI-phase of the cell cycle. In contrast, the concurrent activation of MAPK and PKB/Akt by EGF treatment led to an inhibition of proliferation in SK-BR-3 and can be a ttributed to missing EGFR/c-erbB2 heterodimers. HRG was a strong stimulator of proliferation in both cell lines. Conclusions: We show that in the presence of identical amounts of c-erbB2 r eceptors, the ligand-induced cellular response differs significantly. These differences were mediated by variances in signal transduction, most likely due to different recruitment of heterotypic receptor complexes. Overall, t here is strong evidence that c-erbB2 receptor overexpression in breast canc er cells is an insufficient marker to determine cellular response in terms of cell proliferation. 2001. Cytometry 44:338-348, 2001. (C) 2001 Wiley-Lis s, Inc.