Activation of an EGFR/neu chimeric receptor: early intracellular signals and cell proliferation responses

A. Pandiella, H. Lehvaslaiho, M. Magni, K. Alitalo, J. Meldolesi

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Two clones of NIH3T3 fibroblasts, NEN37 and NEN7, overexpressing chimeric EGF/neu receptors (3 x 105 and 1 x 106 receptors/cell, respectively), were treated with EGF in order to identify the array of intracellular signals generated after activation of the neu proto-oncogene product. The results thus obtained were correlated with the effects of EGF on cell growth, investigated by both [3H]thymidine incorporation and long term (5 days) proliferation studies. In addition to the stimulation of the neu tyrosine kinase, previously reported by Lahvaslaiho et al. (EMBO J., 8, 159-166, 1989), EGF (10-9-10-8 M) was found to induce marked increases of both [Ca2+](i) and plasma membrane potential (investigated by the fura-2 and bis-oxonol techniques) which, in their initial phase, were only marginally dependent on the presence of Ca2+ in the incubation medium. These responses were inhibited, but only in part (40-50%) by phorbol ester activators of protein kinase C. Moreover, inositolphosphate analysis (by anion exchange chromatography) revealed hydrolysis of membrane polyphosphoinositides. All these effects of EGF were more prompt and much larger in NEN7 than NEN37 cells. The EGF concentration-dependence curves (measured by both [3H]thymidine incorporation and long-term proliferation assay) were quite different in the two cell clones. In the cells expressing the lower number of receptors measurable growth stimulation was observed at 10-10, and maximal effect at 10-9 M EGF. In NEN7 cells the curve was much more shallow, with measurable stimulation already at 10-12 and maximal effect at 10-8 M EGF. The maximal growth effect was approximately the same for the two cell clones. It is concluded that the intracellular signals identified here may play a limited role in the neu-induced cell proliferation, but are possibly involved in the acquisition of the tumoral phenotype typically expressed by the EGF-treated NEN7 cells.

Original languageEnglish
Pages (from-to)1299-1305
Number of pages7
Issue number11
Publication statusPublished - 1989

ASJC Scopus subject areas

  • Cancer Research
  • Genetics
  • Molecular Biology


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