Unrepaired clustered DNA lesions induce chromosome breakage in human cells

Abstract

Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis.

Fig. 1.

Clustered DNA damage induced by Fe ions but not by Si ions is refractory to repair. (A Upper) Representative images show recruitment and retention of 53BP1, EGFP-XRCC1, and hOGG1 at the sites of DNA damage induced by 1 Gy of Si or Fe ions. (A Lower) Venn diagrams show percentage of individual and colocalized 53BP1, EGFP-XRCC1, and hOGG1 foci at indicated times after irradiation. Data shown are the percentage of foci relative to the number in cell irradiated for 30 min (the sum of 53BP1, EGFP-XRCC1, and hOGG1 foci numbers at 30 min was defined as 100%). (B) H₂O₂ does not induce clustered DNA damage and γ-rays induce only a small number of clustered DNA lesions. (Upper) Representative images show induction of 53BP1, EGFP-XRCC1, and hOGG1 foci in cells treated with 25 mM H₂O₂ or 1 Gy γ-rays at 30 min after exposure. (Lower) Venn diagrams show percentages of colocalized foci (calculated as in A). HT1080 cells stably expressing EGFP-XRCC1 were treated with different DNA damaging agents, immunostained with anti-53BP1 and hOGG1 antibodies, and the images were recorded using confocal microscopy. In each experiment, the average of individual and colocalized foci in 100–120 cells from two to three independent experiments was used for the calculation. More details are provided in SI Text).

Fig. 2.

Spatial distribution of DSBs, SSBs, and base lesions within the complex DNA damage determines the cellular ability to repair complex DNA damage. (A) 53BP1, XRCC1, and hOGG1 markers colocalize along the dense ionizing tracks traversed by Si and Fe ions. Representative 2D and 3D deconvoluted images show colocalization of 53BP1, XRCC1, and hOGG1 in cells treated with 1 Gy of Si or Fe ions. (B) 53BP1, XRCC1, and hOGG1 markers are tightly colocalized in Fe ion-irradiated cells. Graphs show levels of colocalization of different DNA lesion markers at 10 min and 24 h after 1 Gy of Si or Fe irradiation. Colocalization shown is the average obtained from 20 to 30 cells. Error bars represent SEM. *P = 0.0001, **P = 0.2.3E-09, ***P = 3.5E-10, ****P = 1.4E-12, *****P = 3.8E-13. (C) Fe ions induce very tight spatial distributions of DSB, SSB, and base lesions within the clustered DNA damage site. Representative illustration showing spatial colocalization of DSB, SSB, and damaged base markers within the clustered DNA damage induced by Si and Fe ions. (D) Not all persistent DNA lesions are associated with heterochromatic regions. Representative images show colocalization of 53BP1 with heterochromatic regions (Tri-Me) at indicated times after 1 Gy of Si or Fe ion irradiation. (E) The graph shows the number of 53BP1 foci that were juxtaposed with heterochromatic regions at indicated times after 1 Gy of γ-ray, Si, and Fe irradiation. The average number of colocalized foci in 100–120 cells from three independent experiments was used for the calculation. Error bars represent SEM. Detailed legend is provided in SI Text.

Fig. 3.

Unrepaired clustered DNA lesions result in high levels of gross-chromosomal aberrations in Fe ion-irradiated cells. (A) The total number of gross chromosomal aberrations per mitotic cell. (B) The number of different types of chromosomal aberrations per mitotic cell after 1 Gy of γ-ray, O, Si, and Fe irradiation. Exponentially growing HT1080 cells were irradiated with 1 Gy of radiation, and 16 h later the chromosome preparations were made by accumulating mitotic cells in the presence of 0.1 mg/mL colcemid for 8 h. For each radiation type, more than 100 metaphase spreads were counted. Each data point in the graph is the average of three independent experiments. Error bars represent SD.

Fig. 4.

Checkpoint release before the completion of clustered DNA damage repair represents a major cause for chromosome aberration formation. (A) Representative histograms show cell cycle profile at indicated times after mock treatment or 1 Gy of γ-ray, Si, or Fe irradiation. (B) The graph shows percentage of cells in G2/M phase at indicated times after mock-irradiation or 1 Gy of γ-ray, Si, or Fe irradiation. (C) The graph shows percentage of M phase cells relative to mock-irradiated cells at indicated times. (D) The graph shows percentage of BrdU-positive cells in G2 and (E). G1 phase cells at indicated times after mock-irradiation or treatment with 1 Gy of γ-rays or Si or Fe ions. For D and E, exponentially growing HT1080 cells were labeled with BrdU for 30 min and then irradiated with 1 Gy of Fe, Si, or O ions, or γ-rays. Subsequently, cells were immunostained with FITC-conjugated anti-BrdU antibody and then subjected to flow cytometry. (F) G2/M checkpoint release occurs before the completion of clustered DNA damage repair. Venn diagrams show number of individual and colocalized foci at indicated times after 1 Gy of Fe ion irradiation. More than 120 cells per time point from three independent experiments were examined. More details are provided in SI Text.