Effects of Photon Radiation on DNA Damage, Cell Proliferation, Cell Survival, and Apoptosis of Murine and Human Mesothelioma Cell Lines

Abstract

Purpose: To characterize the cellular responses of murine and human mesothelioma cell lines to different doses of photon radiation with a long-term aim of optimizing a clinically relevant in vivo model in which to study the interaction of radiation therapy and immunotherapy combinations.

Methods and materials: Two murine mesothelioma cell lines (AB1 and AE17) and 3 human cell lines (BYE, MC, and JU) were used in the study. Cells were treated with increasing doses of photon radiation. DNA damage, DNA repair, cell proliferation, and apoptosis at different time points after irradiation were quantified by flow cytometry, and cell survival probability was examined using clonogenic survival assay.

Results: DNA damage increased with escalating dose in all cell lines. Evident G2/M arrest and reduced cell proliferation were observed after irradiation with 8 Gy. DNA repair was uniformly less efficient at higher compared with lower radiation-fraction doses. The apoptosis dose response varied between cell lines, with greater apoptosis observed at 16 Gy with human BYE and murine AB1 cell lines but less for other studied cell lines, regardless of dose and time. The α/β ratio from the cell survival fraction of human mesothelioma cell lines was smaller than from murine ones, suggesting human cell lines in our study were more sensitive to a change of dose per fraction than were murine mesothelioma cell lines. However, in all studied cell lines, colony formation was completely inhibited at 8 Gy.

Conclusions: A threshold dose of 8 Gy appeared to be appropriate for hypofractionated radiation therapy. However, the radiation therapy doses between 4 and 8 Gy remain to be systematically analyzed. These observations provide an accurate picture of the in vitro response of mesothelioma cell lines to photon irradiation and characterize the heterogeneity between human and murine cell lines. This information may guide in vivo experiments and the strengths and limitations of extrapolation from murine experimentation to potential human translation.

Figure 1

DNA damage increases as a function of dose. Murine and human mesothelioma cell lines were treated with increasing doses of photon radiation, whereupon DNA damage responses (γ-H2AX) were quantified by flow cytometry. A-E, DNA damage responses of AB1, AE17, BYE, JU, and MC cell lines, respectively. F, Differences in DNA damage responses specifically with an ED50 (50% DNA damage) parameter between BYE and MC cell lines and AB1 and MC cell lines. One-way analysis of variance for multiple group comparison with Tukey adjustment for pairwise comparison. A 4-parameter log-logistic multiple regression model for DNA damage dose-response analysis. *P < .05; **P < .01; ***P < .001.

Figure 2

High-dose photon radiation arrests the cell cycle of murine cell lines at the G2/M phase. A, B, G2/M phase arrest of the AB1 and AE17 cell lines after 24 hours at 8, 16, and 32 Gy. C-E, cell-cycle pattern of BYE, JU, and MC cell lines. Data are generated from 3 independent repeats.

Figure 3

Global DNA repair responses after radiation therapy are similar for both murine and human mesothelioma cell lines. A-E, DNA repair of AB1, AE17, BYE, MC, and JU cell lines, respectively, after receiving different doses of radiation. F, A comparison of global DNA repair of murine and human mesothelioma cell lines. Analysis of covariance for multiple linear regression model with interaction using Tukey adjustment: the linear model for global γ-H2AX with interaction was visualized as a mean. Adjusted R² is one of the indications of goodness-of-fit of the final model.

Figure 4

Cell proliferation is greatly inhibited by 8-Gy radiation. A-E, Cell proliferation of AB1, AE17, BYE, JU, and MC cell lines, respectively. F, Cell proliferation of the murine and human cell line after exposing to 0, 2, 8, and 16 Gy at 72 hours, fitted using multiple generalized linear least square regression.

Figure 5

Decreasing cell-survival fraction of murine and human mesothelioma cell lines. A, Survival fraction of murine, AB1, and AE17 and B, survival fraction of human mesothelioma cell lines and BYE and JU cell lines.

Figure 6

Apoptosis levels after radiation therapy differ between murine and human mesothelioma cell lines. A-E, Patterns of apoptosis of AB1, AE17, BYE, and MC cells, respectively. F, Comparison of the level of apoptosis among murine and human cell lines with 16 Gy at 72 hours. Data are presented as mean ± standard error. One-way analysis of variance for multiple group comparison with Tukey adjustment for pairwise comparison.