Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activationand enhance radiation toxicity of human mammary carcinoma cells

Exposure of MDA-MB-231 human mammary carcinoma cells to an ionizing radiati on dose of 2 Gy results in immediate activation and Tyr phosphorylation of the epidermal growth factor receptor (EGFR), Doxycycline induced expression of a dominant negative EGFR-CD533 mutant, lacking the COOH-terminal 533 am ino acids, in MDA-TR15-EGFR-CD533 cells was used to characterize intracellu lar signaling responses following irradiation. Within 10 min, radiation exp osure caused an immediate, transient activation of mitogen activated protei n kinase (MAPK) which was completely blocked by expression of EGFR-CD533, T he same radiation treatment also induced an immediate activation of the c-J un-NH2-terminal kinase 1 (JNK1) pathway that was followed by an extended ri se in kinase activity after 30 min. Expression of EGFR-CD533 did not block the immediate JNK1 response but completely inhibited the later activation. Treatment of MDA-TR15-ECFR-CD533 cells with the MEK1/2 inhibitor, PD98059, resulted in similar to 70% inhibition of radiation-induced MAPK activity, a nd potentiated the radiation-induced increase of immediate JNK1 activation twofold. Inhibition of Ras farnesylation with a concomitant inhibition of R es function completely blocked radiation-induced MAPK( and JNK1 activation, Modulation of EGFR and MAPK functions also altered overall cellular respon ses of growth and apoptosis, Induction of EGFR-CD533 or treatment with PD98 059 caused a 3-5-fold increase in radiation toxicity in a novel repeated ra diation exposure growth assay by interfering with cell proliferation and po tentiating apoptosis, In summary, this data demonstrates that both MAPK and JNK1 activation in response to radiation occur through EGFR-dependent and -independent mechanisms, and are mediated by signaling through Ras, Further more, we have demonstrated that radiation-induced activation of EGFR result s in downstream activation of MAPK which may affect the radiosensitivity of carcinoma cells.