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High-Throughput Proton Irradiations Uncover a Differential DNA Damage Repair Response to P53 Depletion in Two Lung Cancer Cell Lines

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L Bronk

L Bronk*, F Guan , D Ma , Y Wang , M Kerr , D Patel , U Titt , O Vassiliev , S Lin , R Mohan , D Grosshans , UT MD Anderson Cancer Center, Houston, TX


SU-K-108-8 (Sunday, July 30, 2017) 4:00 PM - 6:00 PM Room: 108

Purpose: Mutation of the tumor suppressor p53 is often associated with advantages in cellular survival and thus increased therapeutic resistance. We aim to assess the effect of p53 deletion on the cellular response to proton radiation.

Methods: A multi-tiered range shifter was designed using Monte Carlo (MC) simulation to evenly sample proton linear energy transfer (LET). Stable p53-knockout (KO) in A549 and H460 NSCLC cells utilizing the CRISPR/Cas9 platform was confirmed by Western blot. The parental and p53-KO cells were irradiated with a spot-scanning beam. Cells were fixed at multiple timepoints post-irradiation and stained for 53BP1, a DNA double-strand break (DSB) marker, using standard immunofluorescence techniques.

Results: The MC simulations calculated LETs between 1 and 20keV/um within the columns of the irradiation setup. p53-KO cells were found to be more radioresistant when irradiated with Cs-137. For a given LET, both A549 and H460 p53-KO cells were found to have increased DSB repair constants compared to the corresponding wildtype cells. A trend of increasing persistent foci at 24hrs post-irradiation was observed with LET for all lines. The number of terminal foci was reduced in A549-KO cells for all LETs except the lowest value of 1.0keV/um. In contrast, H460-KO cells exhibited decreased persistent foci for low LET protons until the threshold value of 4.2keV/um. At LETs above 4.2keV/um, the number of persistent foci dramatically increased.

Conclusion: Our results indicate that p53 deletion affects the cellular response to proton radiation in the H460 and A549 cell lines in fundamentally different ways. H460 p53-KO cells exhibit a dramatic decrease in DSB repair following proton irradiation that is not observed in wildtype H460 or A549 lines. These results warrant confirmational studies in additional cell lines. Further investigation will be aimed towards identifying the mechanism of action resulting in this proton-energy dependent divergence in DSB repair.

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