Purpose: Under the presence of DNA damage, the DNA repair protein ATR has essential roles in arresting cells in the G2 phase of the cell cycle so that DNA repair can take place. ATR is also a key protein in homologous recombination double strand break repair. Here we investigate the consequences of disrupting cell cycle arrest at G2 through ATR inhibition on cancer cell lines irradiated with photons and protons.
Methods: H1299 and PANC-1 cancer cell lines were treated with DMSO (control), or an ATR inhibitor (ATRi) (AZD6738, 0.1 or 1 µM) for 1 h prior to irradiation. Cells were exposed to 5 Gy of 6 MV x-rays or 9.9 keV/µm protons (dose-weighted LET in water), and then fixed and stained for flow cytometry analysis to evaluate cell cycle distribution (propidium iodide), mitosis (phosho-histone 3) and DNA damage (γ-H2AX). Clonogenic cell survival assays were also performed.
Results: The relative biological effectiveness (RBE) had a non-significant increase for protons+ATRi compared to protons alone. Irradiated cells showed a pronounced G2 arrest after both photons and protons. ATRi 1 µM was effective in abrogating G2 arrest in cells exposed to photons or protons. A higher amount of mitotic cells was observed with 1 µM ATRi+5 Gy compared to DMSO+5 Gy (P-value <0.01). This effect was higher for protons alone compared to photons alone (P-value< 0.01). Persistent γ-H2AX foci was higher for ATRi treated cells compared to DMSO indicating that ATRi also suppressed DNA double strand break repair.
Conclusion: Both photons and protons induced robust cell cycle arrest at G2. Administration of ATRi disrupted the G2 arrest and increased the amount of cells in mitosis. Our results suggest that ATRi is effective at inducing sensitivity of cancer cells to radiation in part because of the role of ATRi on cell cycling.
Funding Support, Disclosures, and Conflict of Interest: This research was supported in part by RP170040, 1R21CA252411-01, Emerson Collective, and MD Anderson Cancer Center. Dr. Sawakuchi has research agreements with Alpha Tau Medical and Artios Pharma. Dr. Shaitelman has a research agreement with Exact Sciences.