The Characteristics Of P53 Pulses Induced By Ionizing Radiation With Different Dosages And Fractionations And Its Biologicaleffects

Objective:This paper is focused on p53 pulses after the different single dose irradiation and two times irradiaton with different time interval, and kinetics characteristics of p53 pulses of human breast cancer cell line MCF-7, human lung adenocarcinoma cancer cell line A549 and normal human fibroblasts cell line NFC, and measuring preliminarily the number、period and amplitude of p53 pulses,and its biological effect.It would be helpful to study damped p53 pulse in dose irradiation segmentation, and explore new way of segmentation model for clinical dose optimization.Methods:Logarithmic growth phase cells were plated in removable 96 well cell culture plates (corning 9102) with 8 rows 12 columns after 24 h, and exposed to respectively 0 gy,2 gy,4 gy,8 gy and 2*2 gy gamma irradiation, p53 protein expression levels were measured by the Cell-based Colorimetric ELISA in 96 well Cell culture plate within 30 h after irradiation;Twice irradiation time at,respectively,8h,12 h and 24 h after the first irraditon.By flow cytometry, and plate cloning experiments,we measured the cell cycle distribution, apoptosis rate, survial fraction of those cells, at different time points before and after irradiation。Result:1. different dosages1.1 With the help of 8 rows 12 columns removable 96 well Cell culture plate, and the Cell-based Colorimetric ELISA method, the damped p53 pulses successfully were measured, to provide a new thought for this research of protein dynamics1.2 At the population level,the characteristic of damped p53 pulses of three kinds of cells included:①Similarities:the p53 pulse number did not depend on radiation dose,and persisted for detection time testing the period and were round 6; the period of p53 pulses were relatively fixed from 3 to 4 hours, the amplitude of p53 pulses depended on radiation dose.②Difference:The pulse peak time is different, the first pulse peak time of NFC cell lines were at 1 h after irradiation, MCF-7 and A549 cell lines were at 3 h after irradiation; the pulse amplitude is different, he results showed that the pulse amplitude ranked from high to low as follows:MCF-7, A549,NFC.1.3 Within 30h after various single doses of y-rays, The first peak time of the apoptosis of MCF-7 and A549 cells occurred at 24 h after irradiation, The peak time of the apoptosis of normal human fibroblasts NFC occurred at 12h after irradiation; The G2/M arrest peak time of those cells occurred at 12 after irradiation。2. different fractionations2.1 After two 2Gy doses of γ-rays with different time intervals (8h、12h、 24h), there were no change in the amplitude and period of damped p53 pulses, interval.compared with the single 2 Gy dose, the amplitude and period of dampde pulse were not obvious changes(P> 0.05); compared with the single 4 Gy, P53 attenuation amplitude of the pulse amplitude is lower than the corresponding time points, the period has not been altered obviously.2.2 At 12h and 24h After single 4Gy or two 2Gy doses of γ-rays with different time intervals (8h、12h、24h), NFC cell lines between groups G2/M phase cells and apoptosis percent were no significant difference (P> 0.05). The G2/M percent of MCF-7 and A549 cell lines between groups were difference (P<0.001), suggesting dose dependent; At 24h after irradiation, The G2/M percent of groups between different time intervals has no obvious difference (P>0.05). Compared groups with two 2Gy doses of γ-rays with different time intervals, the apoptosis rate of the single 4 Gy doses of γ-rays group decreased at 12 h after irradiation, showing spilt-dose effect.3. those cells clone forming efficiency gradually in creased with the time.Conclusion:1. Under conditions of this experiment, the p53 pulse amplitude of those cells are obviously dose dependent, and is aslo associated with cell types, the pulse amplitude ranked from high to low as follows:MCF-7, A549,NFC cell lines.2.the peak time of 53 pulses are significantly different between the tumor and normal cells, the former is at 3 hours after irradiation, the latter is at 1 hour after irradiation. While,the apoptosis peak show obvious consistency.3.Under conditions of this experiment, P53 pulses also appear obvious attenuation model in the fractionated protocol (2 gy* 2 f),and cell types.is correlative with colony formation and cell apoptosis rate.