Study On The Radiobiological Effects Of Cell Surface Receptor EGFR And The Related Signal Transduction Pathway MAPK On Cell Reactions Following Low Dose Ionizing Irradiation

Radioimmunotherapy(RIT), as an option of radiotherapy, plays a role in cancer treatment, but its cure effect in many cases is not delectable. In order to optimize the dosing of RIT, we proposed a new strategy as to divide the drug into mutiple dosage, and we termed it as multiple low dose RIT (MLD-RIT). Our experiments showed that, compared with the traditional RIT, MLD-RIT had better effects in tumor control. Our experiment also suggested that low dose and low dose rate ionizing irradiation (IR) possessed special radiobiological features, some of which have been distinguished by other researchers. But the researchers always waved too much weight on DNA, so neglected cell membrane in their works. So we have the following objects:1. To investigate the effect of low dose IR on EGFR (epithelia growth factor receptor) , which is located on cell surface , and on its related signal trans-duction pathway MAPK (mitogen activated protein kinase).2. To investigate the relationship between EGFR/MAPK and radiosensitivity when cells are exposed to low dose IR.3. To investigate the function of EGFR/MAPK in cell reaction regulations after low dose IR and elucidate further the mechamisms underlying induced senescense we had observed in former experiments.The study of the phosphorylation of EGFR and activation of MAPK induced by low dose IR. We chose A549 for its high expression of EGFR and selected two low dose 2Gy & 6Gy . We detected the level of p-EGFR and p-ERK by western blot, and did the kinase assay of MAPK before and afer IR. We blocked the EGFR/MAPK passway with AG1478 and PD98059 to observe the changes of p-EGFR and MAPK. Our study showed that the level of p-EGFR was elevated right after IR, and reached the maximum 5 minutes after the expo-sion. Similarly, the activity of MAPK was inducedby IR, and the elevation remained a long time after that. Compared with 6Gy group , the level of p-EGFR and the activity of MAPK were lower than that of 2Gy group. In our study, we also observed that AG1478 and PD98059 inhibited both the basal and induced activity by IR.EGFR/MAPK pathway and radiosensitivity in low dose IR. Like the former part of our experiment, we still used A549 . 2Gy & 6Gy, as well as AG1478 and PD98059 to block EGFR/MAPK. We double stained the cells with annexin V and PI to detect apoptosis with flow cytometry. We drawed the growth curves based on the MTT values. When compared the surviving fractions , apoptotic rates and proliferative potential between cells with or without EGFR/MAPK blockage, we obtained the following findings: the blockage reduced the surviving fractions, and the sensitizing factors achieved by AG1478 and PD98059 in 2Gy & 6Gy groups were 1.94&2.26, 1.09&1.10 respectively; these two agents elevated the apoptotic rate of 6Gy group from 1.51 to 3.14 and 3.01; the growth curve of 2Gy & 6Gy groups manifested different slopes after IR: IR slowed down the proliferation rate of the cells in 2Gy group, while totally inhibited the proliferation and even reduced the cell numbers for 6Gy group. AG1478 and PD98059 made the prolifation rate in 2Gy group and cell numbers in 6Gy group even less. It seemed that the two agents had greater effect on the curve of 2Gy group. To study the funtion of EGFR/MAPK pathway in G2 arrest and DNA repair. We continued to use A549 , 2Gy & 6Gy, as well as AG1478&PD98059 to block EGFR/MAPK. We adopted PI-FCM , comet assay and RT-PCR to detect correspondingly G2 cells , DNA damage as well as the mRNA of ERCCU XRCC1, two of DNA repair genes. As the results of the blockage of EGFR/MAPK, cells were arrested in G2 more slowly; the level of DNA damage at the 60 minutes post-IR was elevated, although the onset level was not affected significantly as show by the statistics; as to ERCC1&XRCC1, their mRNA level were elevated after IR, while the blockage inhibited the elevation.To study the relationship between EGFR/MAPK/P21 and endoreplication and induced senescense occurred after low dose IR. Unlike the former experiments, weadopted two cel...