Radiation-induced up-regulation of DNA repair occurs through EGFR/ERK signalling: discovery of a radio-refractory period following the initial irradiation
Medical College of Virginia, Virginia Commonwealth University, Virginia Commonwealth
Aim: We have previously shown that through an ERK1/2 dependent mechanism TGFa is shed from DU145 cells in response to ionizing radiation. Once shed, soluble TGFa confers sustained ERK1/2 activation on un-irradiated bystander cells, resulting in the up-regulation of DNA repair proteins and enhanced tumor growth. Here, we characterize the radiation-induced release of TGFa, reporting for the first time a refractory period following irradiation. Methods: Following exposure of DU145 prostate carcinoma cells to 60Co g-irradiation, TGFa release was measured by ELISA assay. Expression of the DNA repair proteins XRCC1 and ERCC1 was determined by semi-quantitative RT-PCR and Western analyses. Western analysis was also used to examine PARP cleavage, levels of phospho-ERK, and activated EGFR. EGFR activation was inhibited by the tyrphostin AG1478. ERK1/2 activation was blocked by the MEK-specific inhibitor PD98059. Results: Blockade of the EGFR by the tyrphostin AG1478 or blockade of MEK1/2 by PD98059 eliminates the up-regulation XRCC1 and ERCC1 by TGFa, and substantially reduces the up-regulation of both proteins by irradiation. A similar blockade is induced by the initial exposure to ionizing radiation. Following irradiation cells are refractory to further stimulation by irradiation for 8-12 hr. Irradiation during this refractory period does not result in increased expression of the DNA repair proteins but is associated with a marked increase in the apoptotic fraction and cleavage of PARP. During the refractory period, however, the EGFR is subject to ligand-activation. Conclusions: These results show the role of radiation-induced EGFR transactivation in the mediation of TGFa shedding and the up-regulation of certain DNA repair proteins. Following this signalling dependent up-regulation of DNA repair, cells are refractory to additional irradiation, which fails to transactivate the EGFR to induce DNA repair proteins or induce additional shedding of TGFa. More importantly, the appearance of the refractory period heralds a switch in cellular radiation response from DNA repair to apoptosis. We are currently studying the specific EGFR phosphorylation patterns associated with ligand activation and radiation-induced transactivation.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 994 (Signaling pathways - Part II).