Exposure to galactic cosmic radiation compromises DNA repair and increases the potential for oncogenic chromosomal rearrangement in bronchial epithelial cells

Abstract

Participants in deep space missions face protracted exposure to galactic cosmic radiation (GCR). In this setting, lung cancer is a significant component of the overall risk of radiation-exposure induced death. Here we investigate persistent effects of GCR exposure on DNA repair capacity in lung-derived epithelial cells, using an enzyme-stimulated chromosomal rearrangement as an endpoint. Replicate cell cultures were irradiated with energetic ⁴⁸Ti ions (a GCR component) or reference γ-rays. After a six-day recovery, they were challenged by expression of a Cas9/sgRNA pair that creates double-strand breaks simultaneously in the EML4 and ALK loci, misjoining of which creates an EML4-ALK fusion oncogene. Misjoining was significantly elevated in ⁴⁸Ti-irradiated populations, relative to the baseline rate in mock-irradiated controls. The effect was not seen in γ-ray irradiated populations exposed to equal or higher radiation doses. Sequence analysis of the EML4-ALK joints from ⁴⁸Ti-irradiated cultures showed that they were far more likely to contain deletions, sometimes flanked by short microhomologies, than equivalent samples from mock-irradiated cultures, consistent with a shift toward error-prone alternative nonhomologous end joining repair. Results suggest a potential mechanism by which a persistent physiological effect of GCR exposure may increase lung cancer risk.

Figure 1

Experimental strategy and example data. (A) Schematic depicting sites of Cas9/sgRNA cleavage relative to ALK and EML4 genes. Chevrons depict direction of transcription. Cleavage sites are within introns. Exon (ex) numbers are shown. P1 and P2 are primers for Taqman PCR assays. Taqman hybridization probe is shown with fluorophore (green) and quencher (black) that separate upon hybridization to amplicon. (B) Reconstruction experiment showing linearity of Taqman qPCR assay. (C) Experimental timeline showing irradiation, recovery, Cas9/sgRNA challenge, and harvesting of DNA for analysis. (D,E) Example Taqman PCR data. Independent cultures were irradiated, challenged, and DNA collected for analysis. Amplification curves are shown for single-copy internal standard (RNase P, Panel D) or for EML4-ALK junctions (Panel E). Irradiation was performed at an LET of 108 keV/μm). Parallel cultures not challenged with Cas9/sgRNAs were included in the experiment in Panel E but showed no detectable amplification products after 40 cycles of amplification. Plots depict normalized fluorescence reporter values (ΔRn) as a function of cycle number. Green line denotes software-determined threshold for determination of amplification (C_t) values.

Figure 2

Effect of irradiation on the frequency of Cas9/sgRNA-induced EML4-ALK rearrangement. (A) Effect of 108 keV/μm or 200 keV/μm ⁴⁸Ti on response to Cas9/sgRNA challenge. Triplicate cultures were irradiated as indicated, challenged by transduction with lentiviral Cas9/sgRNA vectors, and DNA was analysed by Taqman qPCR using RNase P as an internal standard. Significance was evaluated by ANOVA followed by 2-sided Dunnett t-tests *P < 0.05. (B) Same as panel A but irradiation with ¹³⁷Cs γ-rays. Differences were non-significant. Experiments in panel A and B were performed contemporaneously at the NASA Space Radiation Laboratory or a ¹³⁷Cs irradiator, both of which are located within the Brookhaven National Laboratory.

Figure 3

TIDE analysis of Cas9/sgRNA-stimulated EML4-ALK junctions. (A) Representative primary sequence data. Top panel, amplification of cloned DNA representing exact joining of Cas9/sgRNA-generated blunt ends. Bottom panel, amplification of pooled genomic DNA from a flask of HBEC-3KT F25F cells irradiated with 1.0 Gy of 108 keV/μm ⁴⁸Ti ions and subjected to Cas9/sgRNA challenge. (B) TIDE analyses of DNA from multiple independent cell populations. Samples are from the same experiment as in Fig. 2A. Red bars denote recurrent deletions; grey bars denote exact joining. Only peaks scored as significant are shown. Contribution of each species to the total is indicated (%). Percentages sum to less than 100 because non-significant peaks are not scored or shown.

Figure 4

Sequence analysis of cloned junction sequence DNA. (A) Summary plot showing percentages of clones of different types recovered from different treatment groups. Note concordance with TIDE analysis in Fig. 3B. (B) Proposed origin of sequence deletions. Top sequence shows exact joining, other sequences show recurrent deletions. Deletions are assumed to occur by 3′ resection, followed by resection of unpaired sequence flaps (denoted by scissors icon). Microhomologies are boxed. Some clones contained point mutations as shown. No individual clone had more than one point mutation, and no mutation was recovered more than once in a given deletion context.