P37 Induction of DNA Double Strand Breaks in Human Primary Lung Cells

Abstract

DNA double strand breaks (DSB) are the most detrimental effects on cells by which ionizing radiation (IR) causes cancer. The frequency of DSB induced decreases with time, indicative of DSB repair. One of the earliest responses to DSB damage is the recruitment of the repair protein, 53BP1, to DSB sites. Fluorescent-tagged antibodies specific for 53BP1 can therefore be exploited to detect DSB lesions as discrete ‘fluorescent foci’. This project aimed to assess the frequency of induction and kinetics of DNA DSB repair in cells exposed to IR. Primary human bronchial epithelial cells were cultured, irradiated and fixed at different time-points. Radiation induced DNA DSB were identified by immunofluorescence technique using 53BP1 as a marker protein. Images were captured and analyzed by fluorescence microscope and axiovision software. After exposure to 2Gy radiation, the highest number of foci was visible within 10min (6.613foci/nucleus). The score at 10min in unirradiated and IR cells were significantly different (P=0.00001). After then the trend declined. Average number of 53BP1 foci decreased up to 6hr (2.83 foci/nucleus at 6hr). The score at 10min was statistically different from that of 6hr (P=0.0008). Data stayed the same from 6hr to 16hr (P at 6hr=0.552, at 8hr=0.615 and at 16hr=0.768). After 16hr the foci reduced up to 24hr where it returned to nearly baseline but significant variation remained from that of unirradiated cells at 24hr-point (P=0.001). In case of unirradiated cells, difference was not observed over the whole duration (P>0.05). Initially, after irradiation, a wide variation existed among the proportion of small, medium and large foci which was, however, getting closer with time, and by 24hr it appeared like a cluster of proportion where all fractions of foci remained the same. In unirradiated cells, an average of 1 DSB was seen in each nucleus for a 24hr duration whereas radiation of a dose of 2Gy produced as many as 6 DSB within 10min. DSB were quickly repaired for the first 6hr, and more than half of DSB got repaired within this period. Few DNA lesions persisted even after 24hr. Changes in the proportional size distribution of foci may be indicative of migration and clustering of radiation-induced foci representing slow DSB repair, leading to chance of chromosomal rearrangement, and subsequently increase the risk of cancer cell formation. If this hypothesis is valid and we can locate these clustering time-points specifically, specific time-points of transforming cancer cells after irradiation may be identified.