Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines

Abstract

Background: Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them.

Methods: A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients.

Results: The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h.

Conclusion: So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.

Figure 1

Levels of H4K20me1, H4K20me2 and H4K20me3 in extracts of HuKo LCLs after irradiation with 0 Gy, 2 Gy and 10 Gy and incubation for 15 min, 1 h and 24 h. A) representative Western Blot, Tubulin alpha served as loading control. B) Quantitative evaluation, each normalized to unirradiated controls. Indicated are mean and standard error of the mean from 2–3 independent experiments and 1–2 blots per experiment.

Figure 2

Levels of H3K9me1, H3K9me2 and H3K9me3 in extracts of HuKo LCLs after irradiation with 0 Gy, 2 Gy and 10 Gy and incubation for 15 min, 1 h and 24 h. A) representative Western Blot, Tubulin alpha served as loading control. B) Quantitative evaluation, each normalized to unirradiated controls. Indicated are mean and standard error of the mean from 2–3 independent experiments and 1–3 blots per experiment. ** statistically significant with 0.005 > α > 0.0005.

Figure 3

Levels of H3K27me3 in extracts of HuKo LCLs after irradiation with 0 Gy, 2 Gy and 10 Gy and incubation for 15 min, 1 h and 24 h. A) representative Western Blot, Tubulin alpha served as loading control. B) Quantitative evaluation, each normalized to unirradiated controls. Indicated are mean and standard error of the mean from 2–3 independent experiments and 1–2 blots per experiment.

Figure 4

Levels of unmodified H3K4, H3K4me1, H3K4me2 and H3K4me3 in extracts of HuKo LCLs after irradiation with 0 Gy, 2 Gy and 10 Gy and incubation for 15 min, 1 h and 24 h. A) representative Western Blot, Tubulin alpha served as loading control. B) Quantitative evaluation, each normalized to unirradiated controls. Indicated are mean and standard error of the mean from 2–3 independent experiments and 1–3 blots per experiment. ** statistically significant with 0.005 > α > 0.0005, *** statistically significant with 0.0005 > α > 0.00005.

Figure 5

Levels of H4K16ac, H4K5ac, H3K56ac and H3K9ac in extracts of HuKo LCLs after irradiation with 0 Gy, 2 Gy and 10 Gy and incubation for 15 min, 1 h and 24 h. A) representative Western Blot, Tubulin alpha served as loading control. B) Quantitative evaluation, each normalized to unirradiated controls. Indicated are mean and standard error of the mean from 2–3 independent experiments and 1–2 blots per experiment. * statistically significant with 0.05 > α > 0.005, ** statistically significant with 0.005 > α > 0.0005, *** statistically significant with 0.0005 > α > 0.00005.

Figure 6

Levels of histone modifications H3K56ac, H4K5ac and H4K16ac after irradiation with 10 Gy and incubation for 1 h and 24 h in extracts from radioresistant LUCY LCLs 20037 and 4064, radiosensitive LUCY LCLS 4008, 40028, 36011, and 4060, as well as LCLs from ATM proficient and ATM deficient individuals. Data from HuKo LCLs are shown for comparison. Indicated are means and standard errors of the mean from 2 independent experiments and 2–8 blots per data point. * statistically significant with 0.05 > α > 0.005, ** statistically significant with 0.005 > α > 0.0005, *** statistically significant with 0.0005 > α > 0.00005.