Reardon D B, Contessa J N, Mikkelsen R B, Valerie K, Amir C, Dent P, Schmidt-Ullrich R K
Department of Radiation Oncology, Massey Cancer Center, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia, VA 23298, USA.
Oncogene. 1999 Aug 19;18(33):4756-66. doi: 10.1038/sj.onc.1202849.
Exposure of MDA-MB-231 human mammary carcinoma cells to an ionizing radiation 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 amino acids, in MDA-TR15-EGFR-CD533 cells was used to characterize intracellular signaling responses following irradiation. Within 10 min, radiation exposure caused an immediate, transient activation of mitogen activated protein kinase (MAPK) which was completely blocked by expression of EGFR-CD533. The same radiation treatment also induced an immediate activation of the c-Jun-NH2-terminal kinase 1 (JNK1) pathway that was followed by an extended rise 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-EGFR-CD533 cells with the MEK1/2 inhibitor, PD98059, resulted in approximately 70% inhibition of radiation-induced MAPK activity, and potentiated the radiation-induced increase of immediate JNK1 activation twofold. Inhibition of Ras farnesylation with a concomitant inhibition of Ras function completely blocked radiation-induced MAPK and JNK1 activation. Modulation of EGFR and MAPK functions also altered overall cellular responses of growth and apoptosis. Induction of EGFR-CD533 or treatment with PD98059 caused a 3-5-fold increase in radiation toxicity in a novel repeated radiation exposure growth assay by interfering with cell proliferation and potentiating 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. Furthermore, we have demonstrated that radiation-induced activation of EGFR results in downstream activation of MAPK which may affect the radiosensitivity of carcinoma cells.
将MDA-MB-231人乳腺癌细胞暴露于2 Gy的电离辐射剂量下会导致表皮生长因子受体（EGFR）立即激活并发生酪氨酸磷酸化。在MDA-TR15-EGFR-CD533细胞中，强力霉素诱导表达缺乏COOH末端533个氨基酸的显性负性EGFR-CD533突变体，用于表征辐射后细胞内信号转导反应。在10分钟内，辐射暴露导致丝裂原活化蛋白激酶（MAPK）立即短暂激活，而EGFR-CD533的表达完全阻断了这种激活。相同的辐射处理还诱导了c-Jun-NH2-末端激酶1（JNK1）途径的立即激活，随后在30分钟后激酶活性持续升高。EGFR-CD533的表达并未阻断JNK1的立即反应，但完全抑制了随后的激活。用MEK1/2抑制剂PD98059处理MDA-TR15-EGFR-CD533细胞，导致辐射诱导的MAPK活性约70%受到抑制，并使辐射诱导的JNK1立即激活增加两倍。用伴随抑制Ras功能的法尼基化抑制剂抑制Ras，完全阻断了辐射诱导的MAPK和JNK1激活。EGFR和MAPK功能的调节也改变了细胞的整体生长和凋亡反应。在一种新型的重复辐射暴露生长试验中，EGFR-CD533的诱导或用PD98059处理通过干扰细胞增殖和增强凋亡，使辐射毒性增加3至5倍。总之，这些数据表明，辐射诱导的MAPK和JNK1激活通过EGFR依赖性和非依赖性机制发生，并由Ras信号介导。此外，我们已经证明，辐射诱导的EGFR激活导致MAPK的下游激活，这可能影响癌细胞的放射敏感性。
