Changes in cell growth and viability of HLEC1 following irradiation with sham or <i>D</i>₁₀ of X-rays.

<p>(A) Growth curve. (B) Viability evaluated with a trypan blue dye exclusion assay. Data are presented as means and SDs of three independent experiments with single measurements, where CPD at the time of irradiation was 14.0 ± 0.3 and dose rate was 0.42 ± 0.01 Gy/min.</p

Effect of a continuous treatment with ATMi and/or DNA-PKi on temporal kinetics of 53BP1 focus formation at 0.1, 0.5, 1, 2, 8, 24, 48, 96 or 144 h after 1 Gy irradiation.

<p>(A) HLEC1 and WI-38 after sham irradiation. (B) HLEC1 after 1 Gy irradiation. (C) WI-38 after 1 Gy irradiation. For HLEC1 and WI-38, CPD at the time of irradiation was 14.1 ± 0.3 and 39.9 ± 0.4, and dose rate was 0.44 ± 0.00 and 0.43 ± 0.00 Gy/min, respectively. Inhibitors were added at 0.5–1 h prior to irradiation and continued to exist until cells were fixed at indicated times postirradiation. Data represent means and SDs of three independent experiments, where 100–318 cells were counted for each sample.</p

Ionizing radiation response of primary normal human lens epithelial cells

<div><p>Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.</p></div

Alterations in SA-β-gal positivity.

<p>(A) HLEC1. (B) WI-38 and WI-38/hTERT. CPD shown for HLEC1 at CPD 17.1 ± 0.0 and WI-38/hTERT at CPD 685 ± 0.4 is at the time of plating, and SA-β-gal positivity shown is at 7 days after plating. CPD shown for WI-38 at CPD 68.1 ± 0.0, and WI-38/hTERT at CPD 361 ± 0.1 is at the time of plating, and SA-β-gal positivity shown is at 8 days after plating. CPD shown for HLEC1 at CPD 11.4 ± 0.1 and 14.3 ± 0.1 and WI-38 at CPD 35.1 ± 0.4 is at the time of irradiation with 1, 2, <i>D</i>₁₀ or 8 Gy (at dose rate of 0.44 ± 0.01, 0.43 ± 0.00 and 0.43 ± 0.01 Gy/min, respectively), and SA-β-gal positivity shown is at 7 days postirradiation. Data represent means and SDs of three independent experiments, where 192–2048 cells were counted for each sample. *These data were taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181530#pone.0181530.ref058" target="_blank">58</a>].</p

Effect of a continuous or a 16 h treatment with ATMi and/or DNA-PKi on SA-β-gal positivity at 7 days after <i>D</i>₁₀ irradiation.

<p>(A) A continuous treatment of HLEC1. CPD at the time of irradiation was 14.3 ± 0.1, and dose rate was 0.43 ± 0.00 Gy/min. (B) A 16 h treatment of HLEC1. CPD at the time of irradiation was 11.4 ± 0.1, and dose rate was 0.44 ± 0.01 Gy/min. (C) A continuous treatment of WI-38. CPD at the time of irradiation was 35.1 ± 0.2, and dose rate was 0.43 ± 0.00 Gy/min. (D) A 16 h treatment of WI-38. CPD at the time of irradiation was 34.2 ± 0.2, and dose rate was 0.44 ± 0.00 Gy/min. For a continuous treatment, inhibitors were present from 0.5–1.5 h prior to irradiation until fixation at 7 days postirradiation. For a 16 h treatment, inhibitors were present from 0.25–2.5 h prior to irradiation until 16–17 h after irradiation, and were absent until fixation at 7 days postirradiation. Data are presented as means and SDs of three independent experiments, where 100–1298 cells were counted for each sample.</p

Effect of a continuous or a 16 h treatment with ATMi and/or DNA-PKi on the plating efficiency and survival after 2 Gy irradiation.

<p>(A) Plating efficiency. (B) SF2. Cells were replated for colony formation at 16–17 h after 2 Gy, and colonies were formed for 13 days. For a continuous treatment, inhibitors were present from 1–2.5 h prior to irradiation until fixation of colonies. For a 16 h treatment, inhibitors were present from 0.25–2.5 h prior to irradiation until 16–17 h postirradiation, but were absent during colony formation. For a continuous treatment of HLEC1, CPD at the time of plating was 11.2 ± 0.0, and dose rate was 0.45 ± 0.00 Gy/min. For a continuous treatment of WI-38, CPD at the time of plating was 38.7 ± 0.3. For a 16 h treatment of WI-38, CPD at the time of plating was 35.4 ± 0.2, and dose rate was 0.44 ± 0.00 Gy/min. Data shown are means and SDs of three to ten independent experiments with quadruplicate to quadecuplicate measurements.</p

Representative images of metaphase chromosomes in HLEC1 at CPD 14.5 (A) and WI-38 at CPD 44.7 (B).

<p>Bars, 20 µm.</p

The impact of cell numbers in clonogenic colonies on the surviving fraction in HLEC1 (left panel) and WI-38 (right panel).

<p>The general “colony number-based” surviving fraction was calculated from the number of clonogenic colonies divided by the number of plated cells with correction for the plated efficiency. This was compared here with the “cell number-based” surviving fraction that was calculated as the sum of the integrated density of clonogenic colonies (i.e., cell numbers in clonogenic colonies given a linear relationship between the integrated density and cell numbers in each clonogenic colony) divided by the number of plated cells with correction for that at 0 Gy. Colony number-based survival curves (open symbols with solid lines) were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098154#pone-0098154-g003" target="_blank">Figure 3</a>, and the data of the integrated density of clonogenic colonies presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098154#pone.0098154.s005" target="_blank">Figure S5</a> were used to obtain cell number-based curves (closed symbols with dotted lines). The data represent means and SD of three independent experiments with quadruplicate measurements. *0.01≤<i>p</i><0.05 compared between two types of survival curves at each dose. For WI-38, the difference between the colony number-based survival curve and the cell number-based survival curve was insignificant for all 5 dose points tested (<i>p</i>>0.10), indicating that the colony number-based survival fraction is akin to the cell number-based one. For HLEC1, the difference between two types of survival curves reached a statistical significance only at 4 Gy (<i>p</i> = 0.02), but there was clearly a tendency toward the increased surviving fraction at 2 Gy and 6 Gy.</p

Surviving fraction related to the Bcl-2 effect calculated by the saturation-corrected and adaptive-response methods.

<p>The corresponding experimental data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114056#pone.0114056-Hamada2" target="_blank">[13]</a> obtained from Eq. (8) are also plotted.</p

Karyotype.

<p>Karyotype.</p