Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving

Abstract

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

FIG. 1

RKO36 cell fates. Panel A: The RKO36 HR substrate consists of 1 kbp direct repeats, comprising EGFP coding (GFP) and poly(A) sequences, and a neo resistance marker. One EGFP is driven by the CMV promoter but inactivated by an XhoI linker frameshift (FS) mutation, and the second lacks a promoter. GFP⁺ recombinants can arise by GC, which eliminates the XhoI mutation, or by SSA or crossovers that delete one GFP and the intervening sequence. These outcomes were distinguished by PCR with primers 1, 2 and 3. Panel B: Schematic of time-lapse and end-point DHR assays. Pure GFP⁻ populations produced by FACS were irradiated (or mock irradiated) and surviving cells were seeded to dishes for visualization for seven days by time-lapse microscopy; resultant colonies were also scored in the end-point assay. For each condition, separate dishes were incubated in parallel, and after seven days these cells were harvested, and GFP⁻ cells were generated by FACS and analyzed by time-lapse and end-point assays. This procedure was then repeated for a third week. Colonies arising one week postirradiation were expanded and genomic DNA was prepared for PCR analysis to distinguish GC and SSA products. The sorted GFP⁺ cells can be used to study delayed mutation (to GFP⁻) but this was not performed in this study. Panel C: Individual frames of time-lapse movies show examples of immediate HR (all GFP⁺) and DHR (mixed GFP^+/−) colonies at indicated times; yellow arrows indicate GFP⁺ cells arising in the middle frame. Immediate HR and DHR cells that subsequently senesced or died are shown in the lower three rows.

FIG. 2

Exposure dose-response curves. RKO36 cells were irradiated with X rays, carbon ions and survival was measured by clonogenic assay. Data are averages (±SD) for three replicates per determination.

FIG. 3

Frequencies of carbon-ion irradiation induced immediate HR (IHR) and IHR to senescence or death. Phenotypes were determined by time-lapse microscopy with 97–1,659 initial cells scored per condition (average number of cells scored = 730). Values are averages (+SEM) for 3–4 determinations and statistics were calculated by t tests. *P < 0.05; **P < 0.01.

FIG. 4

X-ray-induced DHR. DHR was scored by end-point assay, with 2–4 dishes monitored and 201–2,606 colonies scored per condition (average number of colonies scored = 1,080). Statistics calculated by Fisher exact tests. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 5

Carbon-ion-induced DHR measured by time-lapse and endpoint assays. Time-lapse values were determined as described in Fig. 3 legend, with 74–1,659 initial cells scored per condition (average number of cells scored = 353). End-point values and statistics were determined as described in Fig. 4 legend, with 488–1,814 colonies scored per condition (average number of cells scored = 1,165).

FIG. 6

Carbon-ion-induced DHR to senescence or death (panel A) and total senescent or dead cells (panel B). These events were determined by time-lapse assays using the same samples and statistical analyses described in Fig. 5 legend.