Assessment of Targeted and Non-Targeted Responses in Cells Deficient in <i>ATM</i> Function following Exposure to Low and High Dose X-Rays

<div><p>Radiation sensitivity at low and high dose exposure to X-rays was investigated by means of chromosomal aberration (CA) analysis in heterozygous <i>ATM</i> mutation carrier and A-T patient (biallelic <i>ATM</i> mutation) lymphoblastoid cell lines (LCLs). Targeted and non-targeted responses to acutely delivered irradiation were examined by applying a co-culture system that enables study of both directly irradiated cells and medium-mediated bystander effects in the same experimental setting. No indication of radiation hypersensitivity was observed at doses of 0.01 Gy or 0.1 Gy for the <i>ATM</i> mutation carrier LCL. The A-T patient cells also did not show low-dose response. There was significant increase in unstable CA yields for both <i>ATM</i> mutation carrier and A-T LCLs at 1 and 2 Gy, the A-T cells displaying more distinct dose dependency. Both chromosome and chromatid type aberrations were induced at an increased rate in the irradiated A-T cells, whereas for <i>ATM</i> carrier cells, only unstable chromosomal aberrations were increased above the level observed in the wild type cell line. No bystander effect could be demonstrated in any of the cell lines or doses applied. Characteristics typical for the A-T cell line were detected, i.e., high baseline frequency of CA that increased with dose. In addition, dose-dependent loss of cell viability was observed. In conclusion, CA analysis did not demonstrate low-dose (≤100 mGy) radiosensitivity in <i>ATM</i> mutation carrier cells or A-T patient cells. However, both cell lines showed increased radiosensitivity at high dose exposure.</p></div

Cell viability in bystander cells.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093211#pone-0093211-g004" target="_blank">Figure 4</a> legend for details.</p

Overview of the experimental design used in the co-culture assay.

<p>In each experiment, LCLs of the same genotype were seeded into 6-well plates (cells exposed to direct irradiation) and insert plates (bystander cells). Cells in 6-well plates (indicated by white circles) were irradiated with X-ray at doses of 0, 0.01, 0.1, 1, or 2 Gy. Immediately after irradiation, inserts with a permeable filter and containing bystander cells (filled black circles) were placed on top of the cells in the 6-well plates. After 1 or 20 hours co-culture, the directly irradiated and bystander cells were separated and incubated for an additional 24 hours before CA assay and 43 hours before cell viability assay were performed. Total post-irradiation time was 44 or 63 hours in the cell viability assay and 25 or 44 hours in the CA assay.</p