DNA Damage and Inflammatory Response of p53 Null H358 Non-Small Cell Lung Cancer Cells to X-Ray Exposure Under Chronic Hypoxia

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Abstract
Hypoxia-induced radioresistance limits therapeutic success in cancer. In addition, p53 mutations are widespread in tumors including non-small cell lung carcinomas (NSCLCs), and they might modify the radiation response of hypoxic tumor cells. We therefore analyzed the DNA damage and inflammatory response in chronically hypoxic (1% O2, 48 h) p53 null H358 NSCLC cells after X-ray exposure. We used the colony-forming ability assay to determine cell survival, γH2AX immunofluorescence microscopy to quantify DNA double-strand breaks (DSBs), flow cytometry of DAPI-stained cells to measure cell cycle distribution, ELISAs to quantify IL-6 and IL-8 secretion in cell culture supernatants, and RNA sequencing to determine gene expression. Chronic hypoxia increased the colony-forming ability and radioresistance of H358 cells. It did not affect the formation or resolution of X-ray-induced DSBs. It reduced the fraction of cells undergoing G2 arrest after X-ray exposure and delayed the onset of G2 arrest. Hypoxia led to an earlier enhancement in cytokines secretion rate after X-irradiation compared to normoxic controls. Gene expression changes were most pronounced after the combined exposure to hypoxia and X-rays and pertained to senescence and different cell death pathways. In conclusion, hypoxia-induced radioresistance is present despite the absence of functional p53. This resistance is related to differences in clonogenicity, cell cycle regulation, cytokine secretion, and gene expression under chronic hypoxia, but not to differences in DNA DSB repair kinetics.
Funding Information
  • Deutsches Zentrum für Luft- und Raumfahrt (FuW 475 Radiation & Hypoxia, 91716300)
  • Higher Education Commission of Pakistan (HEC)—HRDI-UESTP’s/UET’s-Faculty Training in cooperation with the “Deutscher Akademischer Austauschdienst”—German Academic Exchange Service (DAAD) (FuW 475 Radiation & Hypoxia, 91716300)

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