Cytoplasmic irradiation induces mitochondrial-dependent 53BP1 protein relocalization in irradiated and bystander cells

Abstract

The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiation-induced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-rho(0) cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal rho+ cells.

Figure 1

Representative images of HeLa cells stained for 53BP1 immunofluorescence (green) and cytoplasmic DiI staining (red). After fixation, the red cytoplasmic staining appears punctated instead of the more homogenous staining seen in live cells. A, control cells, examples of DiI-stained and unstained cells. B, nuclear targeted irradiation of the DiI-stained cells with 20 helium ions 3 h after irradiation, with an example of a bystander DiI-unstained neighboring cell. Bar, 10 μm.

Figure 2

Fifty percent of the cell population plated on the microbeam dish was DiI stained and mixed with unstained cells. Every cell in the DiI-stained population was microbeam-targeted either through the nucleus or cytoplasm with 20 helium-3 ions. Cells were fixed 1 and 3 h following irradiation, and the number of cells with 53BP1 foci was scored in both DiI-stained and unstained populations (hit and bystander cells, respectively). A, nuclear targeting, 1 h after radiation. B, nuclear targeting, 3 h after radiation. C, cytoplasmic targeting, 1 h after radiation. D, cytoplasmic targeting, 3 h after radiation. *, P < 0.01; **, P < 0.05, compared with control.

Figure 3

Nuclear (A) and cytoplasmic (B) targeting with 1 or 20 helium ions. Cells were fixed 3 h following irradiation. A, half of the cell population DiI stained (grey) or only 1 cell (solid) was targeted through the nucleus, and bystander cells only were scored for 53BP1 foci. B, direct effect (open): the whole cell population was Nile red–stained and targeted through the cytoplasm, and cells were scored for 53BP1 foci; bystander effect (filled): one cell within the population was targeted through the cytoplasm, and nonhit cells were scored for 53BP1 foci. *, P < 0.01; **, P < 0.05, compared with control.

Figure 4

Modulation of induced 53BP1 foci yields. A, five cells within the population were targeted with one helium ion through the nucleus (white) or the cytoplasm (black). DMSO, aminoguanidine (AG), or filipin were added 30 min before radiation and left for the duration of the incubation time. Cells were fixed 3 h following radiation, and the number of 53BP1 foci was scored in bystander cells. *, P < 0.01; **, P < 0.05, compared with untreated bystander NT samples. B, direct cytoplasmic irradiation with one helium ion of every cell in the whole population followed by 3 h incubation in the presence of 1% DMSO (1), 5 μg/mL filipin, or 50 μmol/L aminoguanidine added 30 min before radiation or with 1% DMSO (2) added immediately after radiation. *, P < 0.01, compared with untreated sample.

Figure 5

A, PCR product of the cytochrome b gene in normal ρ⁺ cells and HeLa treated with 250 ng/mL EB for 7 and 16 days. The product is a unique band of 334 bp size. For experiments, cells were used after 10 to 14 days treatment. B, induced foci per cell, cells are fixed 3 h after radiation. Left (bystander effect), five cells within the population of ρ⁺ or pseudo-ρ⁰ are targeted with one helium ion through the nucleus (white) or the cytoplasm (black). Right, direct radiation effect when cytoplasm of every cell are hit by one helium ion. C, induced foci per cell in bystander ρ⁺ and pseudo-ρ⁰ population. Normal ρ⁺ cells and pseudo-ρ⁰ HeLa were cocultured in distinct areas of the microbeam dish. Each cell in one population was targeted either through the nucleus (white) or the cytoplasm only (black), and the second (bystander) population was scored for 53BP1 foci. *, P < 0.01 compared with ρ⁺ sample.

Figure 6

Model for 53BP1 activation after nuclear or cytoplasmic irradiation for direct and bystander cells.